Use of fgfr mutant gene panels in identifying cancer patients that will be responsive to treatment with an fgfr inhibitor

ABSTRACT

Disclosed herein are methods of identifying a cancer patient that will be responsive to treatment with a fibroblast growth factor receptor (FGFR) inhibitor and methods of treating cancer patients. The methods involve evaluating a biological sample from the patient for the presence of one or more FGFR mutants from a FGFR mutant gene panel. Kits and primers for identifying the presence of one or more FGFR mutant genes in a biological sample are also disclosed herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/056,159, filed Sep. 26, 2014, the disclosure of which herebyincorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 6, 2015, isnamed 103693.000781_SL.txt and is 64,689 bytes in size.

TECHNICAL FIELD

Provided herein are methods of identifying a cancer patient that will beresponsive to treatment with a fibroblast growth factor receptorinhibitor and methods of treating the same.

BACKGROUND

The identification of genetic abnormalities can be useful in selectingthe appropriate therapeutic(s) for cancer patients. This is also usefulfor cancer patients failing the main therapeutic option (front-linetherapy) for that cancer type, particularly if there is no acceptedstandard of care for second and subsequent-line therapy. Fibroblastgrowth factor receptors (FGFRs) are a family of receptor tyrosinekinases involved in regulating cell survival, proliferation, migrationand differentiation. FGFR alterations have been observed in somecancers. To date, there are no approved therapies that are efficaciousin patients with FGFR alterations.

SUMMARY

Disclosed herein are methods of identifying a cancer patient that willbe responsive to treatment with a fibroblast growth factor receptor(FGFR) inhibitor comprising: evaluating a biological sample from thepatient for a FGFR mutant from a FGFR mutant gene panel, wherein theFGFR mutant is a FGFR fusion gene or a FGFR single nucleotidepolymorphism, and wherein said evaluating comprises amplifying cDNA witha pair of primers that bind to and amplify one or more FGFR mutants fromthe FGFR mutant gene panel; and determining whether the one or more FGFRmutants from the gene panel are present in the sample, wherein thepresence of the one or more FGFR mutants indicates that the patient willbe responsive to treatment with the FGFR inhibitor.

Also disclosed are methods of treating cancer in a patient comprising:evaluating a biological sample from the patient for the presence of oneor more FGFR mutants from a FGFR mutant gene panel; and treating thepatient with an FGFR inhibitor if one or more FGFR mutants are presentin the sample.

Kits and primers for identifying the presence of one or more FGFR mutantgenes in a biological sample are further provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The summary, as well as the following detailed description, is furtherunderstood when read in conjunction with the appended drawings. For thepurpose of illustrating the disclosed methods, kits, and primers, thereare shown in the drawings exemplary embodiments of the methods, kits,and primers; however, the methods, kits, and primers are not limited tothe specific embodiments disclosed. In the drawings:

FIG. 1 is an illustration of exemplary FGFR fusion genes, the presenceof at least one of which indicates that a patient will be responsive totreatment with an FGFR inhibitor. Also illustrated (small arrows) areexemplary primer locations for amplifying the fusion genes.

FIG. 2, comprising FIGS. 2A-2I, represents Sanger sequencing resultsfrom FFPET samples positive for: A) FGFR3:TACC3 v1; B) FGFR3:TACC3 v3;C) FGFR3:TACC3 Intron; D) FGFR3:BAIAP2L1; E) FGFR2:AFF3; F) FGFR2:BICC1;G) FGFR2:CASP7; H) FGFR2:CCDC6; and I) FGFR2:OFD1.

FIG. 3 illustrates an exemplary strategy for SNP-specific qRT-PCR usinga 3′ dideoxy wild type (WT) blocker oligonucleotide.

FIG. 4 illustrates an exemplary analytical validation strategy fordetecting FGFR SNPs. Experiments were performed on engineered RK3E celllines expressing the FGFR fusions and diluted into a wild type cell lineharboring no FGFR3/FGFR2 fusions.

FIG. 5, comprising FIGS. 5A-5D, illustrates SNP-specific PCR withdideoxy WT blocker for (a) G370C, (B) Y373C, (C) S249C, and (D) R248C.

FIG. 6, comprising FIGS. 6A-6I, represents efficiency standard curvesfor the FGFR fusion gene assays: A) FGFR3:TACC3 v1; B) FGFR3:TACC3 v3;C) FGFR3:TACC3 Intron; D) FGFR3:BAIAP2L1; E) FGFR2:AFF3; F) FGFR2:BICC1;G) FGFR2:CASP7; H) FGFR2:CCDC6; and I) FGFR2:OFD1.

FIG. 7 is an exemplary representation of FGFR fusion gene status inbladder (primary and metastatic), NSCLC (adenocarcinoma and squamous),ovarian, esophageal (primary and metastatic), head and neck (H&N;primary and metastatic), endometrial (metastatic), breast, and prostatecancer.

FIG. 8 is an exemplary representation of FGFR fusion gene and mutationstatus in NSCLC adenocarcinoma and squamous cell carcinoma.

FIG. 9, comprising FIGS. 9A-9D, represents exemplary results from phaseI patient samples. Assays were performed using synthetic template assaycontrol (ST), primers for GAPDH (quality control sample), or primersspecific for: A) FGFR2:BICC1 fusions; B) FGFR3:TACC3 (exon 18:exon 1)fusions; C) FGFR2:CCDC6 fusions; or D) FGFR3:TACC3 v1, FGFR3:TACC3 v3,or FGFR2:CCDC6 fusions. Patient samples are as follows: A—urothelialcarcinoma; B—bladder cancer; C—cholangiocarcinoma; and D—adrenalcarcinoma.

FIG. 10 represents an exemplary Phase I Study design for aFirst-In-Human Study of JNJ-42756493 in patients with advanced solidtumor.

FIG. 11 represents the maximal inhibitory percentage reduction of sum ofthe diameters of targeted lesions from baseline with dose level greaterthan or equal to 6 mg. Solid tumor patients were treated with the FGFRinhibitor JNJ-42756493 at different doses administered either as a dailyregimen or as an intermittent dosing regimen (7 days on-7 days off).Doses and tumor types are indicated. Reduction in tumor was measured asper the RECIST criteria. Patients whose tumors harbor FGFR genetranslocations and mutations appear to be more sensitive to the FGFRinhibitor JNJ-42756493.

FIG. 12 illustrates the expression of various FGFR fusions in RK3E cellsstably transfected with the indicated FGFR fusion.

FIG. 13, comprising FIGS. 13A-13B, illustrates colony formation assaysin RK3E cells stably transfected with the indicated FGFR fusion. (A)0.1% cresyl crystal violet stained 6-well chambers and (B) bar graphillustrating the number of colonies/100 cells plated. Results arerepresentative of two independent experiments.

FIG. 14, comprising FIGS. 14A-14H, illustrates the expression ofexemplary downstream targets in RK3E cells stably transfected with theindicated FGFR fusion.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The disclosed methods, kits, and primers may be understood more readilyby reference to the following detailed description taken in connectionwith the accompanying figures, which form a part of this disclosure. Itis to be understood that the disclosed methods, kits, and primers arenot limited to the specific methods, kits, and primers described and/orshown herein, and that the terminology used herein is for the purpose ofdescribing particular embodiments by way of example only and is notintended to be limiting of the claimed methods, kits, and primers.

Reference to a particular numerical value includes at least thatparticular value, unless the context clearly dictates otherwise. When arange of values is expressed, another embodiment includes from the oneparticular value and/or to the other particular value. Further,reference to values stated in ranges include each and every value withinthat range. All ranges are inclusive and combinable.

It is to be appreciated that certain features of the disclosed methods,kits, and primers which are, for clarity, described herein in thecontext of separate embodiments, may also be provided in combination ina single embodiment. Conversely, various features of the disclosedmethods, kits, and primers that are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany subcombination.

As used herein, the singular forms “a,” “an,” and “the” include theplural.

The following abbreviations are used throughout the specification: FGFR(fibroblast growth factor receptor); LLOQ (lower limit of quantitation);FGFR3:TACC3 (fusion between genes encoding FGFR3 and transforming acidiccoiled-coil containing protein 3); FGFR3:BAIAP2L1 (fusion between genesencoding FGFR3 and brain-specific angiogenesis inhibitor 1-associatedprotein 2-like protein 1); FGFR2:AFF3 (fusion between genes encodingFGFR2 and AF4/FMR2 family, member 3); FGFR2:BICC1 (fusion between genesencoding FGFR2 and bicaudal C homolog 1); FGFR2: CASP7 (fusion betweengenes encoding FGFR2 and caspase 7); FGFR2:CCDC6 (fusion between genesencoding FGFR2 and coiled-coil domain containing 6); FGFR2:OFD1 (fusionbetween genes encoding FGFR2 and oral-facial-digital syndrome 1); FFPET(Formalin-Fixed Paraffin-Embedded Tissue); SNP (single nucleotidepolymorphism); NSCLC (Non-small-cell lung cancer), ct (cycle threshold).

As used herein, “treating” and like terms refer to reducing the severityand/or frequency of cancer symptoms, eliminating cancer symptoms and/orthe underlying cause of said symptoms, reducing the frequency orlikelihood of cancer symptoms and/or their underlying cause, andimproving or remediating damage caused, directly or indirectly, bycancer.

“Biological samples” refers to any sample from a patient in whichcancerous cells can be obtained and RNA can be isolated. Suitablebiological samples include, but are not limited to, blood, lymph fluid,bone marrow, a solid tumor sample, or any combination thereof. In someembodiments, the biological sample can be FFPET.

As used herein, “pre-amplification” refers to a PCR procedure that isperformed prior to the amplifying step in order to increase the quantityof template cDNA for the amplification step. A pre-amplification stepcan be performed, for example, using the TaqMan® PreAmp Master Mix (LifeTechnologies/Applied Biosystems® product #4391128).

As used herein, “amplifying,” “amplify,” and like terms refer to thegeneration of numerous identical copies of a nucleic acid sample.Suitable techniques for amplifying a nucleic acid sample include, butare not limited to, polymerase chain reaction (PCR) and real-timepolymerase chain reaction (RT-PCR). In some embodiments, the amplifyingstep comprises RT-PCR.

FGFR Mutants

As used herein, the phrase “FGFR mutant” refers to a FGFR fusion gene, aFGFR single nucleotide polymorphism, or both.

“FGFR fusion” or “FGFR fusion gene” refers to a gene encoding FGFR(e.g., FGRF2 or FGFR3), or a portion thereof, and one of the hereindisclosed fusion partners, or portion thereof, created by atranslocation between the two genes. The presence of one or more of thefollowing FGFR fusion genes in a biological sample from a patient can bedetermined using the disclosed methods: FGFR3:TACC3 v1, FGFR3:TACC3 v3,FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3,FGFR2:CASP7, FGFR2:CCDC6, FGFR2:OFD1, or any combination thereof. Table1 provides the FGFR fusion genes and the FGFR and fusion partner exonsthat are fused. FIG. 1 provides an illustration of the various FGFRfusion genes. The sequences of the individual FGFR fusion genes aredisclosed in Table 16.

TABLE 1 Fusion Gene FGFR Exon Partner Exon FGFR3:TACC3 v1 18 11FCFR3:TACC3 v3 18 10 FGFR3:TACC3 Intron 18 4 FGFR3:BAIAP2L1 18 2FGFR2:AFF3 19 8 FGFR2:BICC1 19 3 FGFR2:CASP7 19 4 FGFR2:CCDC6 19 2FGFR2:OFD1 19 3

“FGFR single nucleotide polymorphism” (SNP) refers to a FGFR2 or FGFR3gene in which a single nucleotide differs among individuals. Inparticular, FGFR single nucleotide polymorphism” (SNP) refers to a FGFR3gene in which a single nucleotide differs among individuals. Thepresence of one or more of the following FGFR SNPs in a biologicalsample from a patient can be determined using the disclosed methods:FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, FGFR3 Y373C, or any combinationthereof. The sequences of the FGFR SNPs are provided in Table 2.

TABLE 2 FGFR3 mutant Sequence FGFR3 TCGGACCGCGGCAACTACACCTGCGTCGTGGAGAACR248C AAGTTTGGCAGCATCCGGCAGACGTACACGCTGGAC GTGCTGGAG (T)GCTCCCCGCACCGGCCCATCCTGC AGGCGGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGACGCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACAC CCTACGTTACCGTGCTCA (SEQ ID NO: 1)FGFR3 GACCGCGGCAACTACACCTGCGTCGTGGAGAACAAG S249CTTTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGGTGAGGGCCCTGGGGCGGCGCGGGGGTGGGGGCGGCAGTGGCGGTGGTGGTGAGGGAGGGGGTGGCCC CTGAGCGTCATCTGCCCCCACAGAGCGCT (G)CCCG CACCGGCCCATCCTGCAGGCGGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGACGCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACACCCTACGTTACCGTGCTCAAG GTGGGCCACCGTGTGCACGT (SEQ ID NO: 2)FGFR3 GCGGGCAATTCTATTGGGTTTTCTCATCACTCTGCG G370CTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTG GAGGCTGACGAGGCG (T)GCAGTGTGTATGCAGGCA TCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCGCCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGC ACAAGATCTCCCGCTTCCCG (SEQ ID NO: 3)FGFR3 CTAGAGGTTCTCTCCTTGCACAACGTCACCTTTGAG Y373C*GACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCACTCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAG GCGGGCAGTGTGT (G)TGCAGGCATCCTCAGCTACG GGGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCGCCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCC GCTTCCCGCTCAAGC (SEQ ID NO: 4)Sequences correspond to nucleotides 920-1510 of FGFR3 (Genebank ID #NM_000142.4). Nucleotides in bold underline represent the SNP.*Sometimes mistakenly referred to as Y375C in the literature.

As used herein, “FGFR mutant gene panel” includes one or more of theabove listed FGFR mutants. In some embodiments, the FGFR mutant genepanel is dependent upon the patient's cancer type.

The FGFR mutant panel that is used in the evaluating step of thedisclosed methods is based, in part, on the patient's cancer type. Forpatients with bladder cancer, a suitable FGFR mutant gene panel cancomprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1,FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3Y373C, or any combination thereof.

For patients with metastatic bladder cancer, a suitable FGFR mutant genepanel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1,FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3G370C, or FGFR3 Y373C, or any combination thereof.

For patients with ovarian cancer, a suitable FGFR mutant gene panel cancomprise FGFR3:TACC3 v1, FGER3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1,FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3Y373C, or any combination thereof.

For patients with head and neck cancer, a suitable FGFR mutant genepanel can comprise FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR3 R248C, FGFR3S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.

For patients with metastatic head and neck cancer, a suitable FGFRmutant gene panel can comprise FGFR3:BAIAP2L1, FGFR2:CASP7, orFGFR2:OFD1, or any combination thereof.

For patients with esophageal cancer, a suitable FGFR mutant gene panelcan comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR2:BICC1, FGFR2:CASP7,FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or anycombination thereof.

For patients with metastatic esophageal cancer, a suitable FGFR mutantgene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7,FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof.

For patients with non-small-cell lung adenocarcinoma, a suitable FGFRmutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3,FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:AFF3, FGFR2:CASP7, FGFR3R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combinationthereof.

For patients with non-small cell lung squamous cell carcinoma, asuitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6,FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or anycombination thereof.

For patients with metastatic endometrial cancer, a suitable FGFR mutantgene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3Intron, FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR2:CCDC6, or FGFR2:OFD1, or anycombination thereof.

For patients with breast cancer, a suitable FGFR mutant gene panel cancomprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron,FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6, orFGFR2:OFD1, or any combination thereof.

Primers for Amplifying FGFR Mutants

One skilled in the art knows that amplification of nucleic acid requiresprimers that are complementary, and bind to, a 5′ and 3′ region of thenucleic acid strand that flanks the region sought to be amplified. Asused herein, “pair of primers” refers to the forward and reverse primersused in an amplifying step. Pairs of primers suitable for performing thedisclosed methods are listed in Table 3.

TABLE 3 Reverse Forward  Primer Target Primer 5′-3′ FGFR3:TACC3 V1GACCTGGACCGTGTC CTTCCCCAGTTCCAG CTTACC GTTCTT (SEQ ID NO: 5) (SEQ ID NO: 6)  FGFR3:TACC3 V3 AGGACCTGGACCGTG TATAGGTCCGGTGGA TCCTTCAGGG (SEQ ID NO: 7)  (SEQ ID NO: 8)  FGFR3:TACC3 GGCCATCCTGCCCCCGAGCAGTCCAGGTCA Intron (SEQ ID NO: 9)  GCCAG (SEQ ID NO: 10)FGFR3:BAIAP2L1 CTGGACCGTGTCCTT GCAGCCCAGGATTGA ACCGT ACTGT(SEQ ID NO: 11) (SEQ ID NO: 12) FGFR2:BICC1 TGGATCGAATTCTCAGCCAAGCAATCTGCG CTCTCACA TATTTG (SEQ ID NO: 13) (SEQ ID NO: 14)FGFR2:AFF3 TGGTAGAAGACTTGG TCTCCCGGATTATTT ATCGAATTCT CTTCAACA(SEQ ID NO: 15) (SEQ ID NO: 16) FGFR2:CASP7 GCTCTTCAATACAGCACTTGGATCGAATTC CCTGATCA TCACTCTCA (SEQ ID NO: 17) (SEQ ID NO: 18)FGFR2:CCDC6 TGGATCGAATTCTCA GCAAAGCCTGAATTT CTCTCACA TCTTGAATAA(SEQ ID NO: 19) (SEQ ID NO: 20) FGFR2:OFD1 AGGGTGCATCAACTCACTTGGATCGAATTC ATGAATTAG TCACTCTCA (SEQ ID NO: 21) (SEQ ID NO: 22)FGFR3 R248C GCATCCGGCAGACGT CCCCGCCTGCAGGAT ACA (SEQ ID NO: 24)(SEQ ID NO: 23) FGFR3 S249C GCATCCGGCAGACGT CCCCGCCTGCAGGAT ACA(SEQ ID NO: 26) (SEQ ID NO: 25) FGFR3 G370C AGGAGCTGGTGGAGGCCGTAGCTGAGGATG CTGA CCTG (SEQ ID NO: 27) (SEQ ID NO: 28) FGFR3 Y373CCTGGTGGAGGCTGAC AGCCCACCCCGTAGC GAG T (SEQ ID NO: 29) (SEQ ID NO: 30)FGFR3 R248C GTCGTGGAGAACAAG GTCTGGTTGGCCGGC TTTGGC AG (SEQ ID NO: 31)(SEQ ID NO: 32) FGFR3 S249C GTCGTGGAGAACAAG GTCTGGTTGGCCGGC TTTGGC AG(SEQ ID NO: 33) (SEQ ID NO: 34) FGFR3 G370C AGGAGCTGGTGGAGGCCGTAGCTGAGGATG CTGA CCTG (SEQ ID NO: 35) (SEQ ID NO: 36) FGFR3 Y373CGACGAGGCGGGCAGT GAAGAAGCCCACCCC G GTAG (SEQ ID NO: 37) (SEQ ID NO: 38)

Disclosed herein are primers having the nucleic acid sequence of SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or any combination thereof.

Also disclosed herein are sets of primers having the sequences of SEQ IDNO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14,SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ IDNO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:23 andSEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, SEQ ID NO:27 and SEQ IDNO:28, SEQ ID NO:29 and SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, SEQID NO:33 and SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, SEQ ID NO:37and SEQ ID NO:38, or any combination thereof.

In some embodiments, the set of primers can have the sequence of SEQ IDNO:5 and SEQ ID NO:6. In some embodiments, the set of primers can havethe sequence of SEQ ID NO:7 and SEQ ID NO:8. In some embodiments, theset of primers can have the sequence of SEQ ID NO:9 and SEQ ID NO:10. Insome embodiments, the set of primers can have the sequence of SEQ IDNO:11 and SEQ ID NO:12. In some embodiments, the set of primers can havethe sequence of SEQ ID NO:13 and SEQ ID NO:14. In some embodiments, theset of primers can have the sequence of SEQ ID NO:15 and SEQ ID NO:16.In some embodiments, the set of primers can have the sequence of SEQ IDNO:17 and SEQ ID NO:18. In some embodiments, the set of primers can havethe sequence of SEQ ID NO:19 and SEQ ID NO:20. In some embodiments, theset of primers can have the sequence of SEQ ID NO:21 and SEQ ID NO:22.In some embodiments, the set of primers can have the sequence of SEQ IDNO:23 and SEQ ID NO:24. In some embodiments, the set of primers can havethe sequence of SEQ ID NO:25 and SEQ ID NO:26. In some embodiments, theset of primers can have the sequence of SEQ ID NO:27 and SEQ ID NO:28.In some embodiments, the set of primers can have the sequence of SEQ IDNO:29 and SEQ ID NO:30. In some embodiments, the set of primers can havethe sequence of SEQ ID NO:31 and SEQ ID NO:32. In some embodiments, theset of primers can have the sequence of SEQ ID NO:33 and SEQ ID NO:34.In some embodiments, the set of primers can have the sequence of SEQ IDNO:35 and SEQ ID NO:36. In some embodiments, the set of primers can havethe sequence of SEQ ID NO:37 and SEQ ID NO:38. In some embodiments, theset of primers can have the sequences of any combination of the abovesets of primers.

FGFR Inhibitors for Use in the Disclosed Methods

Suitable FGFR inhibitors for use in the disclosed methods are providedherein.

In some embodiments, if one or more FGFR mutants are present in thesample, the patient can be treated with a FGFR inhibitor disclosed inU.S. Publ. No. 2013/0072457 A1 (incorporated herein by reference),including any tautomeric or stereochemically isomeric form thereof, anda N-oxide thereof, a pharmaceutically acceptable salt thereof, or asolvate thereof (suitable R groups are also disclosed in U.S. Publ. No.2013/0072457 A1). In some aspects, for example, the patient can betreated withN-(3,5-dimethoxyphenyl)-N′-(1-methylethyl)-N-[3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl]ethane-1,2-diamine(referred to herein as “JNJ-42756493” or “JNJ493”):

including a N-oxide thereof, a pharmaceutically acceptable salt thereof,or a solvate thereof. In some aspects, the pharmaceutically acceptablesalt is a HCl salt. In some aspects, the patient can be treated withJNJ493 base.

In some embodiments, the patient can be treated with a FGFR inhibitor ifone or more FGFR mutants are present in the sample, wherein the FGFRinhibitor isN-[5-[2-(3,5-Dimethoxyphenyl)ethyl]-2H-pyrazol-3-yl]-4-(3,5-diemthylpiperazin-1-yl)benzamide(AZD4547), as described in Gavine, P. R., et al., AZD4547: An OrallyBioavailable, Potent, and Selective Inhibitor of the Fibroblast GrowthFactor Receptor Tyrosine Kinase Family, Cancer Res. Apr. 15, 2012 72;2045:

including, when chemically possible, any tautomeric or stereochemicallyisomeric form thereof, and a N-oxide thereof, a pharmaceuticallyacceptable salt thereof, or a solvate thereof.

In some embodiments, the patient can be treated with a FGFR inhibitor ifone or more FGFR mutants are present in the sample, wherein the FGFRinhibitor is3-(2,6-Dichloro-3,5-dimethoxy-phenyl)-1-{6-[4-(4-ethyl-piperazin-1-yl)-phenylamino]-pyrimid-4-yl}-1-methyl-urea(NVP-BGJ398) as described in Int'l Publ. No. WO2006/000420:

including, when chemically possible, any tautomeric or stereochemicallyisomeric form thereof, and a N-oxide thereof, a pharmaceuticallyacceptable salt thereof, or a solvate thereof.

In some embodiments, the patient can be treated with a FGFR inhibitor ifone or more FGFR mutants are present in the sample, wherein the FGFRinhibitor is4-amino-5-fluoro-3-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-quinolin-2-one(dovitinib) as described in Int't Publ. No. WO2006/127926:

including, when chemically possible, any tautomeric or stereochemicallyisomeric form thereof, and a N-oxide thereof, a pharmaceuticallyacceptable salt thereof, or a solvate thereof.

In some embodiments, the patient can be treated with a FGFR inhibitor ifone or more FGFR mutants are present in the sample, wherein the FGFRinhibitor is6-(7-((1-Aminocyclopropyl)-methoxy)-6-methoxyquinolin-4-yloxy)-N-methyl-1-naphthamide(AL3810) (lucitanib; E-3810), as described in Bello, E. et al., E-3810Is a Potent Dual Inhibitor of VEGFR and FGFR that Exerts AntitumorActivity in Multiple Preclinical Models, Cancer Res Feb. 15, 201171(A)1396-1405 and Int'l Publ. No. WO2008/112408:

including, when chemically possible, any tautomeric or stereochemicallyisomeric form thereof, and a N-oxide thereof, a pharmaceuticallyacceptable salt thereof, or a solvate thereof.

In some embodiments, the patient can be treated with a FGFR inhibitor ifone or more FGFR mutants are present in the sample, wherein the FGFRinhibitor is an anti-FGFR2 antibody such as that described inWO2013/076186.

Additional suitable FGFR inhibitors include BAY1163877 (Bayer),BAY1179470 (Bayer), TAS-120 (Taiho), ARQ087 (ArQule), ASP5878(Astellas), FF284 (Chugai), FP-1039 (GSK/FivePrime), Blueprint,LY-2874455 (Lilly), RG-7444 (Roche), or any combination thereof,including, when chemically possible, any tautomeric or stereochemicallyisomeric forms thereof, N-oxides thereof, pharmaceutically acceptablesalts thereof, or solvates thereof.

In some embodiments, the patient can be treated with a FGFR inhibitor ifone or more FGFR mutants are present in the sample, wherein the FGFRinhibitor is BAY1163877 (Bayer), including, when chemically possible,any tautomeric or stereochemically isomeric form thereof, N-oxidethereof, pharmaceutically acceptable salt thereof, or solvate thereof.

In some embodiments, the patient can be treated with a FGFR inhibitor ifone or more FGFR mutants are present in the sample, wherein the FGFRinhibitor is BAY1179470 (Bayer), including, when chemically possible,any tautomeric or stereochemically isomeric form thereof, N-oxidethereof, pharmaceutically acceptable salt thereof, or solvate thereof.

In some embodiments, the patient can be treated with a FGFR inhibitor ifone or more FGFR mutants are present in the sample, wherein the FGFRinhibitor is TAS-120 (Taiho), including, when chemically possible, anytautomeric or stereochemically isomeric form thereof, N-oxide thereof,pharmaceutically acceptable salt thereof, or solvate thereof.

In some embodiments, the patient can be treated with a FGFR inhibitor ifone or more FGFR mutants are present in the sample, wherein the FGFRinhibitor is ARQ087 (ArQule), including, when chemically possible, anytautomeric or stereochemically isomeric form thereof, N-oxide thereof,pharmaceutically acceptable salt thereof, or solvate thereof.

In some embodiments, the patient can be treated with a FGFR inhibitor ifone or more FGFR mutants are present in the sample, wherein the FGFRinhibitor is ASP5878 (Astellas), including, when chemically possible,any tautomeric or stereochemically isomeric form thereof, N-oxidethereof, pharmaceutically acceptable salt thereof, or solvate thereof.

In some embodiments, the patient can be treated with a FGFR inhibitor ifone or more FGFR mutants are present in the sample, wherein the FGFRinhibitor is FF284 (Chugai), including, when chemically possible, anytautomeric or stereochemically isomeric form thereof, N-oxide thereof,pharmaceutically acceptable salt thereof, or solvate thereof.

In some embodiments, the patient can be treated with a FGFR inhibitor ifone or more FGFR mutants are present in the sample, wherein the FGFRinhibitor is FP-1039 (GSK/FivePrime), including, when chemicallypossible, any tautomeric or stereochemically isomeric form thereof,N-oxide thereof, pharmaceutically acceptable salt thereof, or solvatethereof.

In some embodiments, the patient can be treated with a FGFR inhibitor ifone or more FGFR mutants are present in the sample, wherein the FGFRinhibitor is Blueprint, including, when chemically possible, anytautomeric or stereochemically isomeric form thereof, N-oxide thereof,pharmaceutically acceptable salt thereof, or solvate thereof.

In some embodiments, the patient can be treated with a FGFR inhibitor ifone or more FGFR mutants are present in the sample, wherein the FGFRinhibitor is LY-2874455 (Lilly), including, when chemically possible,any tautomeric or stereochemically isomeric form thereof, N-oxidethereof, pharmaceutically acceptable salt thereof, or solvate thereof.

In some embodiments, the patient can be treated with a FGFR inhibitor ifone or more FGFR mutants are present in the sample, wherein the FGFRinhibitor is RG-7444 (Roche), including, when chemically possible, anytautomeric or stereochemically isomeric form thereof, N-oxide thereof,pharmaceutically acceptable salt thereof, or solvate thereof.

Salts can be synthesized from a parent compound that contains a basic oracidic moiety by conventional chemical methods such as methods describedin Pharmaceutical Salts: Properties, Selection, and Use, P. HeinrichStahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8,Hardcover, 388 pages, August 2002, which is incorporated herein byreference. Generally, such salts can be prepared by reacting the freeacid or base forms of these compounds with the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, nonaqueous media such as ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are used. The FGFR inhibitors for use inthe disclosed methods may exist as mono- or di-salts depending upon thepKa of the acid from which the salt is formed.

Acid addition salts may be formed with a wide variety of acids, bothinorganic and organic. Examples of acid addition salts include saltsformed with an acid including, but not limited to, acetic,2,2-dichloroacetic, adipic, alginic, ascorbic (e.g. L-ascorbic),L-aspartic, benzenesulphonic, benzoic, 4-acetamidobenzoic, butanoic, (+)camphoric, camphor-sulphonic, (+)-(1S)-camphor-10-sulphonic, capric,caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulphuric,ethane-1,2-disulphonic, ethanesulphonic, 2-hydroxyethanesulphonic,formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic,glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic),α-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic,isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic,maleic, malic, (−)-L-malic, malonic, (±)-DL-mandelic, methanesulphonic,naphthalenesulphonic (e.g. naphthalene-2-sulphonic),naphthalene-1,5-disulphonic, 1-hydroxy-2-naphthoic, nicotinic, nitric,oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic,L-pyroglutamic, pyruvic, salicylic, 4-amino-salicylic, sebacic, stearic,succinic, sulphuric, tannic, (+)-L-tartaric, thiocyanic,toluenesulphonic (e.g. p-toluenesulphonic), undecylenic and valericacids, as well as acylated amino acids and cation exchange resins.

One particular group of salts consists of salts formed from acetic,hydrochloric, hydriodic, phosphoric, nitric, sulphuric, citric, lactic,succinic, maleic, malic, isethionic, fumaric, benzenesulphonic,toluenesulphonic, methanesulphonic (mesylate), ethanesulphonic,naphthalenesulphonic, valeric, propanoic, butanoic, malonic, glucuronicand lactobionic acids. Another group of acid addition salts includessalts formed from acetic, adipic, ascorbic, aspartic, citric, DL-Lactic,fumaric, gluconic, glucuronic, hippuric, hydrochloric, glutamic,DL-malic, methanesulphonic, sebacic, stearic, succinic and tartaricacids.

If the compound is anionic, or has a functional group which may beanionic (e.g., —COOH may be —COO⁻), then a salt may be formed with asuitable cation. Examples of suitable inorganic cations include, but arenot limited to, alkali metal ions such as Na⁺ and K⁺, alkaline earthmetal cations such as Ca²⁺ and Mg²⁺, and other cations such as Al³⁺.Examples of suitable organic cations include, but are not limited to,ammonium ion (i.e., NH₄ ⁺) and substituted ammonium ions (e.g., NH₃R⁺,NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺).

Examples of some suitable substituted ammonium ions are those derivedfrom: ethylamine, diethylamine, dicyclohexylamine, triethylamine,butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine,benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, aswell as amino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

Where the compounds contain an amine function, these may form quaternaryammonium salts, for example by reaction with an alkylating agentaccording to methods well known to the skilled person. Such quaternaryammonium compounds are within the scope of the disclosed compounds.Compounds containing an amine function may also form N-oxides. Areference herein to a compound that contains an amine function alsoincludes the N-oxide. Where a compound contains several amine functions,one or more than one nitrogen atom may be oxidised to form an N-oxide.Particular examples of N-oxides are the N-oxides of a tertiary amine ora nitrogen atom of a nitrogen-containing heterocycle. N-Oxides can beformed by treatment of the corresponding amine with an oxidizing agentsuch as hydrogen peroxide or a per-acid (e.g. a peroxycarboxylic acid),see for example Advanced Organic Chemistry, by Jerry March, 4^(th)Edition, Wiley Interscience, pages. More particularly, N-oxides can bemade by the procedure of L. W. Deady (Syn. Comm. (1977), 7, 509-514) inwhich the amine compound is reacted with m-chloroperoxybenzoic acid(MCPBA), for example, in an inert solvent such as dichloromethane.

As used herein, the term “solvate” means a physical association of thecompound with one or more solvent molecules. This physical associationinvolves varying degrees of ionic and covalent bonding, includinghydrogen bonding. In certain instances the solvate will be capable ofisolation, for example when one or more solvent molecules areincorporated in the crystal lattice of the crystalline solid. The term“solvate” is intended to encompass both solution-phase and isolatablesolvates. Non-limiting examples of suitable solvates include thedisclosed compounds in combination with water, isopropanol, ethanol,methanol, DMSO, ethyl acetate, acetic acid, ethanolamine and the like.The compound may exert its biological effects while in solution.

Solvates are well known in pharmaceutical chemistry. They can beimportant to the processes for the preparation of a substance (e.g. inrelation to their purification), the storage of the substance (e.g. itsstability) and the ease of handling of the substance, and are oftenformed as part of the isolation or purification stages of a chemicalsynthesis. A person skilled in the art can determine by means ofstandard and long used techniques whether a hydrate or other solvate hasformed by the isolation conditions or purification conditions used toprepare a given compound. Examples of such techniques includethermogravimetric analysis (TGA), differential scanning calorimetry(DSC), X-ray crystallography (e.g. single crystal X-ray crystallographyor X-ray powder diffraction) and Solid State NMR (SS-NMR, also known asMagic Angle Spinning NMR or MAS-NMR). Such techniques are as much a partof the standard analytical toolkit of the skilled chemist as NMR, IR,HPLC and MS. Alternatively the skilled person can deliberately form asolvate using crystallisation conditions that include an amount of thesolvent required for the particular solvate. Thereafter the standardmethods described above, can be used to establish whether solvates hadformed. Also encompassed are any complexes (e.g. inclusion complexes orclathrates with compounds such as cyclodextrins, or complexes withmetals) of the FGFR inhibitor.

Furthermore, the compound may have one or more polymorph (crystalline)or amorphous forms.

The compounds include compounds with one or more isotopic substitutions,and a reference to a particular element includes within its scope allisotopes of the element. For example, a reference to hydrogen includeswithin its scope ¹H, ²H (D), and ³H (T). Similarly, references to carbonand oxygen include within their scope respectively ¹²C, ¹³C and ¹⁴C and¹⁶O and ¹⁸O. The isotopes may be radioactive or non-radioactive. In oneembodiment, the compounds contain no radioactive isotopes. Suchcompounds are preferred for therapeutic use. In another embodiment,however, the compound may contain one or more radioisotopes. Compoundscontaining such radioisotopes may be useful in a diagnostic context.

In some embodiments, the patient is treated with a FGFR inhibitor if oneor more FGFR mutants are present in the sample, wherein the FGFRinhibitor isN-(3,5-dimethoxyphenyl)-N′-(1-methylethyl)-N-[3-(1-methyl-1H-pyrazol-4-yl)quinoxalin-6-yl]ethane-1,2-diamine(referred to herein “JNJ-42756493”), or a pharmaceutically acceptablesalt thereof or a solvate thereof.

Methods of Treating Cancer in a Patient

Disclosed herein are methods of treating cancer in a patient comprising:evaluating a biological sample from the patient for the presence of oneor more FGFR mutants from a FGFR mutant gene panel; and treating thepatient with an FGFR inhibitor if one or more FGFR mutants are presentin the sample.

The disclosed methods can be used to treat a variety of cancer typesincluding, but not limited to, bladder cancer, metastatic bladdercancer, ovarian cancer, head and neck cancer, metastatic head and neckcancer, esophageal cancer, metastatic esophageal cancer, non-small-celllung adenocarcinoma, non-small cell lung squamous cell carcinoma,prostate cancer, lung cancer, gastric cancer, urothelial carcinoma,small cell lung cancer, breast cancer, endometrial cancer, metastaticendometrial cancer, cholangiocarcinoma, hepatocellular carcinoma,glioblastoma, gliomas, colon carcinoma, sarcomas, solid tumors ofsquamous origin, and multiple myeloma.

The FGFR mutant panel that is used in the evaluating step is based, inpart, on the patient's cancer type. For patients with bladder cancer,for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3,FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, orany combination thereof. Accordingly, in some embodiments, a patienthaving bladder cancer is treated with an FGFR inhibitor if FGFR3:TACC3v1 is present in the sample. In some embodiments, a patient havingbladder cancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 ispresent in the sample. In some embodiments, a patient having bladdercancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present inthe sample. In some embodiments, a patient having bladder cancer istreated with an FGFR inhibitor if FGFR2:BICC1 is present in the sample.In some embodiments, a patient having bladder cancer is treated with anFGFR inhibitor if FGFR2:AFF3 is present in the sample. In someembodiments, a patient having bladder cancer is treated with an FGFRinhibitor if FGFR2:CASP7 is present in the sample. In some embodiments,a patient having bladder cancer is treated with an FGFR inhibitor ifFGFR3 R248C is present in the sample. In some embodiments, a patienthaving bladder cancer is treated with an FGFR inhibitor if FGFR3 S249Cis present in the sample. In some embodiments, a patient having bladdercancer is treated with an FGFR inhibitor if FGFR3 G370C is present inthe sample. In some embodiments, a patient having bladder cancer istreated with an FGFR inhibitor if FGFR3 Y373C is present in the sample.In some embodiments, a patient having bladder cancer is treated with anFGFR inhibitor if any combination of the above FGFR mutants is presentin the sample.

For patients with metastatic bladder cancer, for example, a suitableFGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3,FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.Accordingly, in some embodiments, a patient having metastatic bladdercancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present inthe sample. In some embodiments, a patient having metastatic bladdercancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present inthe sample. In some embodiments, a patient having metastatic bladdercancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present inthe sample. In some embodiments, a patient having metastatic bladdercancer is treated with an FGFR inhibitor if FGFR2:BICC1 is present inthe sample. In some embodiments, a patient having metastatic bladdercancer is treated with an FGFR inhibitor if FGFR2:AFF3 is present in thesample. In some embodiments, a patient having metastatic bladder canceris treated with an FGFR inhibitor if FGFR2:CASP7 is present in thesample. In some embodiments, a patient having metastatic bladder canceris treated with an FGFR inhibitor if FGFR3 R248C is present in thesample. In some embodiments, a patient having metastatic bladder canceris treated with an FGFR inhibitor if FGFR3 S249C is present in thesample. In some embodiments, a patient having metastatic bladder canceris treated with an FGFR inhibitor if FGFR3 G370C is present in thesample. In some embodiments, a patient having metastatic bladder canceris treated with an FGFR inhibitor if FGFR3 Y373C is present in thesample. In some embodiments, a patient having metastatic bladder canceris treated with an FGFR inhibitor if any combination of the above FGFRmutants is present in the sample.

For patients with ovarian cancer, for example, a suitable FGFR mutantgene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1,FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in someembodiments, a patient having ovarian cancer is treated with an FGFRinhibitor if FGFR3:TACC3 v1 is present in the sample. In someembodiments, a patient having ovarian cancer is treated with an FGFRinhibitor if FGFR3:TACC3 v3 is present in the sample. In someembodiments, a patient having ovarian cancer is treated with an FGFRinhibitor if FGFR3:BAIAP2L1 is present in the sample. In someembodiments, a patient having ovarian cancer is treated with an FGFRinhibitor if FGFR2:BICC1 is present in the sample. In some embodiments,a patient having ovarian cancer is treated with an FGFR inhibitor ifFGFR2:AFF3 is present in the sample. In some embodiments, a patienthaving ovarian cancer is treated with an FGFR inhibitor if FGFR2:CASP7is present in the sample. In some embodiments, a patient having ovariancancer is treated with an FGFR inhibitor if FGFR3 R248C is present inthe sample. In some embodiments, a patient having ovarian cancer istreated with an FGFR inhibitor if FGFR3 S249C is present in the sample.In some embodiments, a patient having ovarian cancer is treated with anFGFR inhibitor if FGFR3 G370C is present in the sample. In someembodiments, a patient having ovarian cancer is treated with an FGFRinhibitor if FGFR3 Y373C is present in the sample. In some embodiments,a patient having ovarian cancer is treated with an FGFR inhibitor if anycombination of the above FGFR mutants is present in the sample.

For patients with head and neck cancer, for example, a suitable FGFRmutant gene panel can comprise FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR3 R248C,FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.Accordingly, in some embodiments, a patient having head and neck canceris treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in thesample. In some embodiments, a patient having head and neck cancer istreated with an FGFR inhibitor if FGFR2:CASP7 is present in the sample.In some embodiments, a patient having head and neck cancer is treatedwith an FGFR inhibitor if FGFR3 R248C is present in the sample. In someembodiments, a patient having head and neck cancer is treated with anFGFR inhibitor if FGFR3 S249C is present in the sample. In someembodiments, a patient having head and neck cancer is treated with anFGFR inhibitor if FGFR3 G370C is present in the sample. In someembodiments, a patient having head and neck cancer is treated with anFGFR inhibitor if FGFR3 Y373C is present in the sample. In someembodiments, a patient having head and neck cancer is treated with anFGFR inhibitor if any combination of the above FGFR mutants is presentin the sample.

For patients with metastatic head and neck cancer, for example, asuitable FGFR mutant gene panel can comprise FGFR3:BAIAP2L1,FGFR2:CASP7, or FGFR2:OFD1, or any combination thereof. Accordingly, insome embodiments, a patient having metastatic head and neck cancer istreated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in thesample. In some embodiments, a patient having metastatic head and neckcancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present inthe sample. In some embodiments, a patient having metastatic head andneck cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is presentin the sample. In some embodiments, a patient having metastic head andneck cancer is treated with an FGFR inhibitor if any combination of theabove FGFR mutants is present in the sample.

For patients with esophageal cancer, for example, a suitable FGFR mutantgene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR2:BICC1,FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, orany combination thereof. Accordingly, in some embodiments, a patienthaving esophageal cancer is treated with an FGFR inhibitor ifFGFR3:TACC3 v1 is present in the sample. In some embodiments, a patienthaving esophageal cancer is treated with an FGFR inhibitor ifFGFR3:TACC3 v3 is present in the sample. In some embodiments, a patienthaving esophageal cancer is treated with an FGFR inhibitor ifFGFR2:BICC1 is present in the sample. In some embodiments, a patienthaving esophageal cancer is treated with an FGFR inhibitor ifFGFR2:CASP7 is present in the sample. In some embodiments, a patienthaving esophageal cancer is treated with an FGFR inhibitor if FGFR3R248C is present in the sample. In some embodiments, a patient havingesophageal cancer is treated with an FGFR inhibitor if FGFR3 S249C ispresent in the sample. In some embodiments, a patient having esophagealcancer is treated with an FGFR inhibitor if FGFR3 G370C is present inthe sample. In some embodiments, a patient having esophageal cancer istreated with an FGFR inhibitor if FGFR3 Y373C is present in the sample.In some embodiments, a patient having esophageal cancer is treated withan FGFR inhibitor if any combination of the above FGFR mutants ispresent in the sample.

For patients with metastatic esophageal cancer, for example, a suitableFGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3,FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3,FGFR2:CASP7, FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof.Accordingly, in some embodiments, a patient having metastatic esophagealcancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present inthe sample. In some embodiments, a patient having metastatic esophagealcancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present inthe sample. In some embodiments, a patient having metastatic esophagealcancer is treated with an FGFR inhibitor if FGFR3:TACC3 Intron ispresent in the sample. In some embodiments, a patient having metastaticesophageal cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 ispresent in the sample. In some embodiments, a patient having metastaticesophageal cancer is treated with an FGFR inhibitor if FGFR2:BICC1 ispresent in the sample. In some embodiments, a patient having metastaticesophageal cancer is treated with an FGFR inhibitor if FGFR2:AFF3 ispresent in the sample. In some embodiments, a patient having metastaticesophageal cancer is treated with an FGFR inhibitor if FGFR2:CASP7 ispresent in the sample. In some embodiments, a patient having metastaticesophageal cancer is treated with an FGFR inhibitor if FGFR2:CCD6 ispresent in the sample. In some embodiments, a patient having metastaticesophageal cancer is treated with an FGFR inhibitor if FGFR2:OFD1 ispresent in the sample. In some embodiments, a patient having metastaticesophageal cancer is treated with an FGFR inhibitor if any combinationof the above FGFR mutants is present in the sample.

For patients with non-small cell lung (NSCL) adenocarcinoma, forexample, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1,FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:AFF3,FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, orany combination thereof. Accordingly, in some embodiments, a patienthaving NSCL adenocarcinoma is treated with an FGFR inhibitor ifFGFR3:TACC3 v1 is present in the sample. In some embodiments, a patienthaving NSCL adenocarcinoma is treated with an FGFR inhibitor ifFGFR3:TACC3 v3 is present in the sample. In some embodiments, a patienthaving NSCL adenocarcinoma is treated with an FGFR inhibitor ifFGFR3:TACC3 Intron is present in the sample. In some embodiments, apatient having NSCL adenocarcinoma is treated with an FGFR inhibitor ifFGFR3:BAIAP2L1 is present in the sample. In some embodiments, a patienthaving NSCL adenocarcinoma is treated with an FGFR inhibitor ifFGFR2:AFF3 is present in the sample. In some embodiments, a patienthaving NSCL adenocarcinoma is treated with an FGFR inhibitor ifFGFR2:CASP7 is present in the sample. In some embodiments, a patienthaving NSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3R248C is present in the sample. In some embodiments, a patient havingNSCL adenocarcinoma is treated with an FGFR inhibitor if FGFR3 S249C ispresent in the sample. In some embodiments, a patient having NSCLadenocarcinoma is treated with an FGFR inhibitor if FGFR3 G370C ispresent in the sample. In some embodiments, a patient having NSCLadenocarcinoma is treated with an FGFR inhibitor if FGFR3 Y373C ispresent in the sample. In some embodiments, a patient having NSCLadenocarcinoma is treated with an FGFR inhibitor if any combination ofthe above FGFR mutants is present in the sample.

For patients with non-small cell lung (NSCL) squamous cell carcinoma,for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3,FGFR2:CASP7, FGFR2:CCDC6, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, orFGFR3 Y373C, or any combination thereof. Accordingly, in someembodiments, a patient having NSCL squamous cell carcinoma is treatedwith an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. Insome embodiments, a patient having NSCL squamous cell carcinoma istreated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in thesample. In some embodiments, a patient having NSCL squamous cellcarcinoma is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 is presentin the sample. In some embodiments, a patient having NSCL squamous cellcarcinoma is treated with an FGFR inhibitor if FGFR2:BICC1 is present inthe sample. In some embodiments, a patient having NSCL squamous cellcarcinoma is treated with an FGFR inhibitor if FGFR2:AFF3 is present inthe sample. In some embodiments, a patient having NSCL squamous cellcarcinoma is treated with an FGFR inhibitor if FGFR2:CASP7 is present inthe sample. In some embodiments, a patient having NSCL squamous cellcarcinoma is treated with an FGFR inhibitor if FGFR2:CCDC6 is present inthe sample. In some embodiments, a patient having NSCL squamous cellcarcinoma is treated with an FGFR inhibitor if FGFR3 R248C is present inthe sample. In some embodiments, a patient having NSCL squamous cellcarcinoma is treated with an FGFR inhibitor if FGFR3 S249C is present inthe sample. In some embodiments, a patient having NSCL squamous cellcarcinoma is treated with an FGFR inhibitor if FGFR3 G370C is present inthe sample. In some embodiments, a patient having NSCL squamous cellcarcinoma is treated with an FGFR inhibitor if FGFR3 Y373C is present inthe sample. In some embodiments, a patient having NSCL squamous cellcarcinoma is treated with an FGFR inhibitor if any combination of theabove FGFR mutants is present in the sample.

For patients with metastatic endometrial cancer, for example, a suitableFGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3,FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR2:CCDC6, orFGFR2:OFD1, or any combination thereof. Accordingly, in someembodiments, a patient having metastatic endometrial cancer is treatedwith an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. Insome embodiments, a patient having metastatic endometrial cancer istreated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in thesample. in some embodiments, a patient having metastatic endometrialcancer is treated with an FGFR inhibitor if FGFR3:TACC3 Intron ispresent in the sample. In some embodiments, a patient having metastaticendometrial cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1is present in the sample. In some embodiments, a patient havingmetastatic endometrial cancer is treated with an FGFR inhibitor ifFGFR2:CASP7 is present in the sample. In some embodiments, a patienthaving metastatic endometrial cancer is treated with an FGFR inhibitorif FGFR2:CCDC6 is present in the sample. In some embodiments, a patienthaving metastatic endometrial cancer is treated with an FGFR inhibitorif FGFR2:OFD1 is present in the sample. In some embodiments, a patienthaving metastatic endometrial cancer is treated with an FGFR inhibitorif any combination of the above FGFR mutants is present in the sample.

For patients with breast cancer, for example, a suitable FGFR mutantgene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7,FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof. Accordingly, insome embodiments, a patient having breast cancer is treated with an FGFRinhibitor if FGFR3:TACC3 v1 is present in the sample. In someembodiments, a patient having breast cancer is treated with an FGFRinhibitor if FGFR3:TACC3 v3 is present in the sample. In someembodiments, a patient having breast cancer is treated with an FGFRinhibitor if FGFR3:TACC3 Intron is present in the sample. In someembodiments, a patient having breast cancer is treated with an FGFRinhibitor if FGFR3:BAIAP2L1 is present in the sample. In someembodiments, a patient having breast cancer is treated with an FGFRinhibitor if FGFR2:BICC1 is present in the sample. In some embodiments,a patient having breast cancer is treated with an FGFR inhibitor ifFGFR2:AFF3 is present in the sample. In some embodiments, a patienthaving breast cancer is treated with an FGFR inhibitor if FGFR2:CASP7 ispresent in the sample. In some embodiments, a patient having breastcancer is treated with an FGFR inhibitor if FGFR2:CCD6 is present in thesample. In some embodiments, a patient having breast cancer is treatedwith an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In someembodiments, a patient having breast cancer is treated with an FGFRinhibitor if any combination of the above FGFR mutants is present in thesample.

For patients with hepatocellular carcinoma, for example, a suitable FGFRmutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3,FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3,FGFR2:CASP7, FGFR2:CCDC6, FGFR2:OFD1, FGFR3 R248C, FGFR3 S249C, FGFR3G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if FGFR3:TACC3 Intron is present in the sample. Insome embodiments, a patient having hepatocellular carcinoma is treatedwith an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. Insome embodiments, a patient having hepatocellular carcinoma is treatedwith an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if FGFR2:AFF3 is present in the sample. In someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if FGFR2:CASP7 is present in the sample. In someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if FGFR2:CCDC6 is present in the sample. In someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if FGFR2:OFD1 is present in the sample. In someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if FGFR3 R248C is present in the sample. In someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if FGFR3 S249C is present in the sample. In someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if FGFR3 G370C is present in the sample. In someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if FGFR3 Y373C is present in the sample. In someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if any combination of the above FGFR mutants ispresent in the sample.

In some embodiments, the evaluating step comprises: isolating RNA fromthe biological sample; synthesizing cDNA from the isolated RNA;pre-amplifying the cDNA; and amplifying the pre-amplified cDNA with apair of primers that bind to and amplify one or more FGFR mutants fromthe FGFR mutant gene panel.

Isolating RNA from the biological sample can be performed by a number ofprocedures known to one skilled in the art. In one embodiment, RNA canbe isolated from the biological sample using an AllPrep DNA/RNA FFPE Kitfrom Qiagen (product #80234)

Synthesizing cDNA from isolated RNA can be performed by a number ofprocedures known to one skilled in the art. In one embodiment, cDNA canbe synthesized from isolated RNA using a High Capacity cDNA ReverseTranscriptase Kit with RNase Inhibitor from ABI (product #4374966).

Pre-amplification of cDNA can be performed by a number of proceduresknown to one skilled in the art. Amplification procedures are well knownin the art. In one embodiment, cDNA can be pre-amplified using a TaqMan®PreAmp Master Mix (Life Technologies/Applied Biosystems® product#4391128).

In some embodiments, the amplifying step can comprise performingreal-time PCR (qRT-PCR). Exemplary qRT-PCR procedures are discussed inthe Example section herein. In some aspects, the qRT-PCR can be aTaqman® Real-Time PCR assay. qRT-PCR procedures can involve the use ofprobes to increase the specificity of the assay. Suitable probes for usein the qRT-PCR assay include any of the probes disclosed herein, forexample, the probes disclosed in Table 15. In some embodiments, forexample, the real-time PCR can be performed with one or more probescomprising SEQ ID NO: 43, SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO: 46,SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO:51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and/or SEQ ID NO: 55.In other embodiments, the real-time PCR can be performed with one ormore probes consisting essentially of SEQ ID NO: 43, SEQ ID NO:44, SEQID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49,SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO:54, and/or SEQ ID NO: 55. In other embodiments, the real-time PCR can beperformed with one or more probes consisting of SEQ ID NO: 43, SEQ IDNO:44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53,SEQ ID NO: 54, and/or SEQ ID NO: 55. In other embodiments, the real-timePCR can be performed with one or more probes having SEQ ID NO: 43, SEQID NO:44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48,SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO:53, SEQ ID NO: 54, and/or SEQ ID NO: 55.

The qRT-PCR can be performed with one or more 3′ blockingoligonucleotides. Exemplary qRT-PCR procedures using 3′ blockingoligonucleotides are disclosed in the Example section herein. Suitable3′ blocking oligonucleotides include, for example, those disclosed inTable 8. In some embodiments, the qRT-PCR can be performed with one ormore 3′ blocking oligonucleotides comprising SEQ ID NO: 39, SEQ ID NO:40, SEQ ID NO: 41, and/or SEQ ID NO: 42. In some embodiments, theqRT-PCR can be performed with one or more 3′ blocking oligonucleotidesconsisting essentially of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41,and/or SEQ ID NO: 42. In some embodiments, the qRT-PCR can be performedwith one or more 3′ blocking oligonucleotides consisting of SEQ ID NO:39, SEQ ID NO: 40, SEQ ID NO: 41, and/or SEQ ID NO: 42. In someembodiments, the qRT-PCR can be performed with one or more 3′ blockingoligonucleotides having SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41,and/or SEQ ID NO: 42.

Suitable pairs of primers for use in the amplifying step include thosedisclosed in Table 3. For example, in some embodiments, the FGFR mutantand pair of primers can be FGFR3:TACC3 v1 and primers having the aminoacid sequences of SEQ ID NO:5 and SEQ ID NO:6. In some embodiments, theFGFR mutant and pair of primers can be FGFR3:TACC3 v3 and primers havingthe amino acid sequences of SEQ ID NO:7 and SEQ ID NO:8. In someembodiments, the FGFR mutant and pair of primers can be FGFR3:TACC3Intron and primers having the amino acid sequences of SEQ ID NO:9 andSEQ ID NO:10. In some embodiments, the FGFR mutant and pair of primerscan be FGFR3: BAIAP2L1 and primers having the amino acid sequences ofSEQ ID NO:11 and SEQ ID NO:12. In some embodiments, the FGFR mutant andpair of primers can be FGFR2:BICC1 and primers having the amino acidsequences of SEQ ID NO:13 and SEQ ID NO:14. In some embodiments, theFGFR mutant and pair of primers can be FGFR2:AFF3 and primers having theamino acid sequences of SEQ ID NO:15 and SEQ ID NO:16. In someembodiments, the FGFR mutant and pair of primers can be FGFR2:CASP7 andprimers having the amino acid sequences of SEQ ID NO:17 and SEQ IDNO:18. In some embodiments, the FGFR mutant and pair of primers can beFGFR2:CCDC6 and primers having the amino acid sequences of SEQ ID NO:19and SEQ ID NO:20. In some embodiments, the FGFR mutant and pair ofprimers can be FGFR2:OFD1 and primers having the amino acid sequences ofSEQ ID NO:21 and SEQ ID NO:22. In some embodiments, the FGFR mutant andpair of primers can be R248C and primers having the amino acid sequencesof SEQ ID NO:23 and SEQ ID NO:24 or SEQ ID NO:31 and SEQ ID NO:32. Insome embodiments, the FGFR mutant and pair of primers can be S249C andprimers having the amino acid sequences of SEQ ID NO:25 and SEQ ID NO:26or SEQ ID NO:33 and SEQ ID NO:34. In some embodiments, the FGFR mutantand pair of primers can be G370C and primers having the amino acidsequences of SEQ ID NO:27 and SEQ ID NO:28 or SEQ ID NO:35 and SEQ IDNO:36. In some embodiments, the FGFR mutant and pair of primers can beY373C and primers having the amino acid sequences of SEQ ID NO:29 andSEQ ID NO:30 or SEQ ID NO:37 and SEQ ID NO:38. In some embodiments, theFGFR mutant and pair of primers can be any combination of the abovedisclosed FGFR mutants and corresponding pair of primers.

In some embodiments, the amplifying step can be performed with thefollowing:

-   -   a. the pair of primers have the sequences SEQ ID NO:5 and SEQ ID        NO:6 and the probe has the sequence of SEQ ID NO:43;    -   b. the pair of primers have the sequences SEQ ID NO:7 and SEQ ID        NO:8 and the probe has the sequence of SEQ ID NO:44;    -   c. the pair of primers have the sequences SEQ ID NO:9 and SEQ ID        NO:10 and the probe has the sequence of SEQ ID NO:46;    -   d. the pair of primers have the sequences SEQ ID NO:11 and SEQ        ID NO:12 and the probe has the sequence of SEQ ID NO:47;    -   e. the pair of primers have the sequences SEQ ID NO:13 and SEQ        ID NO:14 and the probe has the sequence of SEQ ID NO:45;    -   f. the pair of primers have the sequences SEQ ID NO:15 and SEQ        ID NO:16 and the probe has the sequence of SEQ ID NO:48;    -   g. the pair of primers have the sequences SEQ ID NO:17 and SEQ        ID NO:18 and the probe has the sequence of SEQ ID NO:49;    -   h. the pair of primers have the sequences SEQ ID NO:19 and SEQ        ID NO:20 and the probe has the sequence of SEQ ID NO:50;    -   i. the pair of primers have the sequences SEQ ID NO:21 and SEQ        ID NO:22 and the probe has the sequence of SEQ ID NO:51;    -   j. the pair of primers have the sequences SEQ ID NO:23 and SEQ        ID NO:24 and the probe has the sequence of SEQ ID NO:52;    -   k. the pair of primers have the sequences SEQ ID NO:25 and SEQ        ID NO:26 and the probe has the sequence of SEQ ID NO:53;    -   l. the pair of primers have the sequences SEQ ID NO:27 and SEQ        ID NO:28 and the probe has the sequence of SEQ ID NO:54;    -   m. the pair of primers have the sequences SEQ ID NO:29 and SEQ        ID NO:30 and the probe has the sequence of SEQ ID NO:55;    -   n. the pair of primers have the sequences SEQ ID NO:31 and SEQ        ID NO:32, the probe has the sequence of SEQ ID NO:52, and the 3′        blocking oligonucleotide has the sequence of SEQ ID NO:39;    -   o. the pair of primers have the sequences SEQ ID NO:33 and SEQ        ID NO:34, the probe has the sequence of SEQ ID NO:53, and the 3′        blocking oligonucleotide has the sequence of SEQ ID NO:40;    -   p. the pair of primers have the sequences SEQ ID NO:35 and SEQ        ID NO:36, the probe has the sequence of SEQ ID NO:54, and the 3′        blocking oligonucleotide has the sequence of SEQ ID NO:41;    -   q. the pair of primers have the sequences SEQ ID NO:37 and SEQ        ID NO:38, the probe has the sequence of SEQ ID NO:55, and the 3′        blocking oligonucleotide has the sequence of SEQ ID NO:42; or    -   r. any combination thereof.

The disclosed methods comprise treating a patient if one or more FGFRmutants are present in the sample. The presence of one or more FGFRmutants in the sample can be determined by, for example, sequencing theamplified cDNA.

Suitable FGFR inhibitors for use in the treatment methods include thosepreviously described herein.

Also disclosed are FGFR inhibitors for use in the treatment of cancer ina patient wherein the patient is identified as being responsive totreatment with the FGFR inhibitor by evaluating a biological sampleobtained from the patient for the presence of one or more FGFR mutantsfrom a FGFR mutant gene panel, wherein the presence of the one or moreFGFR mutants in the sample is detected.

Further disclosed are FGFR inhibitors for use in the treatment of cancerin a patient wherein the patient is identified as being responsive totreatment with the FGFR inhibitor by evaluating a biological sampleobtained from the patient for the presence of one or more FGFR mutantsfrom a FGFR mutant gene panel, wherein the one or more FGFR mutants area FGFR fusion gene or a FGFR SNP, wherein the presence of the one ormore FGFR mutants in the sample is detected, and wherein said evaluatingcomprises amplifying cDNA with a pair of primers that bind to andamplify one or more FGFR mutants from the FGFR mutant gene panel.

Further disclosed are FGFR inhibitors for use in the treatment of cancerin a patient wherein the patient is identified as being responsive totreatment with the FGFR inhibitor by evaluating a biological sampleobtained from the patient for the presence of one or more FGFR mutantsfrom a FGFR mutant gene panel, wherein the FGFR mutant is a FGFR fusiongene or a FGFR SNP, wherein the presence of one or more FGFR mutants inthe sample is detected, and wherein said evaluating comprises amplifyinga pre-amplified cDNA with a pair of primers that bind to and amplify oneor more FGFR mutants from the FGFR mutant gene panel.

Methods of Identifying a Cancer Patient that will be Responsive toTreatment with a Fibroblast Growth Factor Receptor (FGFR) Inhibitor

Disclosed herein are methods of identifying a cancer patient that willbe responsive to treatment with a fibroblast growth factor receptor(FGFR) inhibitor comprising: evaluating a biological sample from thepatient for a FGFR mutant from a FGFR mutant gene panel, wherein theFGFR mutant is a FGFR fusion gene or a FGFR single nucleotidepolymorphism, and wherein said evaluating comprises amplifying cDNA witha pair of primers that bind to and amplify one or more FGFR mutants fromthe FGFR mutant gene panel and determining whether the one or more FGFRmutants from the gene panel are present in the sample, wherein thepresence of the one or more FGFR mutants indicates that the patient willbe responsive to treatment with the FGFR inhibitor.

Also provided are methods of identifying a cancer patient that isresponsive to treatment with a fibroblast growth factor receptor (FGFR)inhibitor comprising: evaluating a biological sample from the patientfor a FGFR mutant from a FGFR mutant gene panel, wherein the FGFR mutantis a FGFR fusion gene or a FGFR single nucleotide polymorphism, andwherein said evaluating comprises amplifying cDNA with a pair of primersthat bind to and amplify one or more FGFR mutants from the FGFR mutantgene panel and determining whether the one or more FGFR mutants from thegene panel are present in the sample, wherein the presence of the one ormore FGFR mutants indicates that the patient is responsive to treatmentwith the FGFR inhibitor.

Further provided are methods of identifying a cancer patient that isresponsive to treatment with a fibroblast growth factor receptor (FGFR)inhibitor comprising evaluating a biological sample from the patient forthe presence of one or more FGFR mutant from a FGFR mutant gene panel,wherein the FGFR mutant is a FGFR fusion gene or a FGFR singlenucleotide polymorphism, wherein the presence of the one or more FGFRmutants indicates that the patient is responsive to treatment with theFGFR inhibitor.

In some embodiments, the evaluating can comprise amplifying cDNA with apair of primers that bind to and amplify one or more FGFR mutants fromthe FGFR mutant gene panel. In some embodiments, the cDNA can bepre-amplified cDNA.

In some embodiments, the evaluating step comprises: isolating RNA fromthe biological sample and synthesizing cDNA from the isolated RNA. Insome aspects, the evaluating step can be performed on preamplified cDNA.Thus, the evaluating step can further comprise pre-amplifying the cDNAprior to said amplifying step. Isolating RNA from a biological samplecan be performed by a number of procedures known to one skilled in theart. In one embodiment, RNA can be isolated from the biological sampleusing an AllPrep DNA/RNA FFPE Kit from Qiagen (for example, product#80234). Synthesizing cDNA from isolated RNA can be performed by anumber of procedures known to one skilled in the art. In one embodiment,cDNA can be synthesized from isolated RNA using a High Capacity cDNAReverse Transcriptase Kit with RNase Inhibitor from ABI (for example,product #4374966). Pre-amplification of cDNA can be performed by anumber of procedures known to one skilled in the art. Amplificationprocedures are well known in the art. In one embodiment, cDNA can bepre-amplified using a TaqMan® PreAmp Master Mix (LifeTechnologies/Applied Biosystems® product #4391128).

The disclosed methods can be used to identify patients with a number ofdifferent types of cancer that will be responsive to treatment with afibroblast growth factor receptor (FGFR) inhibitor including, but notlimited to, bladder cancer, metastatic bladder cancer, ovarian cancer,head and neck cancer, esophageal cancer, non-small-cell lungadenocarcinoma, non-small cell lung squamous cell carcinoma, prostatecancer, lung cancer, gastric cancer, urothelial carcinoma, small celllung cancer, breast cancer, endometrial cancer, cholangiocarcinoma,hepatocellular carcinoma, glioblastoma, gliomas, colon carcinoma,sarcomas, solid tumors of squamous origin, and multiple myeloma.

The FGFR mutant panel that is used in the evaluating step is based, inpart, on the patient's cancer type. For patients with bladder cancer,for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3,FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, orany combination thereof. Accordingly, in some embodiments, theevaluating step comprises determining whether FGFR3:TACC3 v1 is presentin a biological sample from a patient having bladder cancer. In someembodiments, the evaluating step comprises determining whetherFGFR3:TACC3 v3 is present in a biological sample from a patient havingbladder cancer. In some embodiments, the evaluating step comprisesdetermining whether FGFR3:BAIAP2L1 is present in a biological samplefrom a patient having bladder cancer. In some embodiments, theevaluating step comprises determining whether FGFR2:BICC1 is present ina biological sample from a patient having bladder cancer. In someembodiments, the evaluating step comprises determining whetherFGFR2:AFF3 is present in a biological sample from a patient havingbladder cancer. In some embodiments, the evaluating step comprisesdetermining whether FGFR2:CASP7 is present in a biological sample from apatient having bladder cancer. In some embodiments, the evaluating stepcomprises determining whether FGFR3 R248C is present in a biologicalsample from a patient having bladder cancer. In some embodiments, theevaluating step comprises determining whether FGFR3 S249C is present ina biological sample from a patient having bladder cancer. In someembodiments, the evaluating step comprises determining whether FGFR3G370C is present in a biological sample from a patient having bladdercancer. In some embodiments, the evaluating step comprises determiningwhether FGFR3 Y373C is present in a biological sample from a patienthaving bladder cancer. In some embodiments, the evaluating stepcomprises determining whether any combination of the above FGFR mutantsare present in a biological sample from a patient having bladder cancer.

For patients with metastatic bladder cancer, for example, a suitableFGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3,FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.Accordingly, in some embodiments, the evaluating step comprisesdetermining whether FGFR3:TACC3 v1 is present in a biological samplefrom a patient having metastatic bladder cancer. In some embodiments,the evaluating step comprises determining whether FGFR3:TACC3 v3 ispresent in a biological sample from a patient having metastatic bladdercancer. In some embodiments, the evaluating step comprises determiningwhether FGFR3:BAIAP2L1 is present in a biological sample from a patienthaving metastatic bladder cancer. In some embodiments, the evaluatingstep comprises determining whether FGFR2:BICC1 is present in abiological sample from a patient having metastatic bladder cancer. Insome embodiments, the evaluating step comprises determining whetherFGFR2:AFF3 is present in a biological sample from a patient havingmetastatic bladder cancer. In some embodiments, the evaluating stepcomprises determining whether FGFR2:CASP7 is present in a biologicalsample from a patient having metastatic bladder cancer. In someembodiments, the evaluating step comprises determining whether FGFR3R248C is present in a biological sample from a patient having metastaticbladder cancer. In some embodiments, the evaluating step comprisesdetermining whether FGFR3 S249C is present in a biological sample from apatient having metastatic bladder cancer. In some embodiments, theevaluating step comprises determining whether FGFR3 G370C is present ina biological sample from a patient having metastatic bladder cancer. Insome embodiments, the evaluating step comprises determining whetherFGFR3 Y373C is present in a biological sample from a patient havingmetastatic bladder cancer. In some embodiments, the evaluating stepcomprises determining whether any combination of the above FGFR mutantsare present in a biological sample from a patient having metastaticbladder cancer.

For patients with ovarian cancer, for example, a suitable FGFR mutantgene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1,FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in someembodiments, the evaluating step comprises determining whetherFGFR3:TACC3 v1 is present in a biological sample from a patient havingovarian cancer. In some embodiments, the evaluating step comprisesdetermining whether FGFR3:TACC3 v3 is present in a biological samplefrom a patient having ovarian cancer. In some embodiments, theevaluating step comprises determining whether FGFR3:BAIAP2L1 is presentin a biological sample from a patient having ovarian cancer. In someembodiments, the evaluating step comprises determining whetherFGFR2:BICC1 is present in a biological sample from a patient havingovarian cancer. In some embodiments, the evaluating step comprisesdetermining whether FGFR2:AFF3 is present in a biological sample from apatient having ovarian cancer. In some embodiments, the evaluating stepcomprises determining whether FGFR2:CASP7 is present in a biologicalsample from a patient having ovarian cancer. In some embodiments, theevaluating step comprises determining whether FGFR3 R248C is present ina biological sample from a patient having ovarian cancer. In someembodiments, the evaluating step comprises determining whether FGFR3S249C is present in a biological sample from a patient having ovariancancer. In some embodiments, the evaluating step comprises determiningwhether FGFR3 G370C is present in a biological sample from a patienthaving ovarian cancer. In some embodiments, the evaluating stepcomprises determining whether FGFR3 Y373C is present in a biologicalsample from a patient having ovarian cancer. In some embodiments, theevaluating step comprises determining whether any combination of theabove FGFR mutants is present in a biological sample from a patienthaving ovarian cancer.

For patients with head and neck cancer, for example, a suitable FGFRmutant gene panel can comprise FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR3 R248C,FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.Accordingly, in some embodiments, the evaluating step comprisesdetermining whether FGFR3:BAIAP2L1 is present in a biological samplefrom a patient having head and neck cancer. In some embodiments, theevaluating step comprises determining whether FGFR2:CASP7 is present ina biological sample from a patient having head and neck cancer. In someembodiments, the evaluating step comprises determining whether FGFR3R248C is present in a biological sample from a patient having head andneck cancer. In some embodiments, the evaluating step comprisesdetermining whether FGFR3 S249C is present in a biological sample from apatient having head and neck cancer. In some embodiments, the evaluatingstep comprises determining whether FGFR3 G370C is present in abiological sample from a patient having head and neck cancer. In someembodiments, the evaluating step comprises determining whether FGFR3Y373C is present in a biological sample from a patient having head andneck cancer. In some embodiments, the evaluating step comprisesdetermining whether any combination of the above FGFR mutants is presentin a biological sample from a patient having head and neck cancer.

For patients with metastatic head and neck cncer, for example, asuitable FGFR mutant gene panel can comprise FGFR3:BAIAP2L1,FGFR2:CASP7, or FGFR2:OFD1, or any combination thereof. Accordingly, insome embodiments, a patient having metastatic head and neck cancer istreated with an FGFR inhibitor if FGFR3:BAIAP2L1 is present in thesample. In some embodiments, a patient having metastatic head and neckcancer is treated with an FGFR inhibitor if FGFR2:CASP7 is present inthe sample. In some embodiments, a patient having metastatic head andneck cancer is treated with an FGFR inhibitor if FGFR2:OFD1 is presentin the sample. In some embodiments, a patient having metastic head andneck cancer is treated with an FGFR inhibitor if any combination of theabove FGFR mutants is present in the sample.

For patients with esophageal cancer, for example, a suitable FGFR mutantgene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR2:BICC1,FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, orany combination thereof. Accordingly, in some embodiments, theevaluating step comprises determining whether FGFR3:TACC3 v1 is presentin a biological sample from a patient having esophageal cancer. In someembodiments, the evaluating step comprises determining whetherFGFR3:TACC3 v3 is present in a biological sample from a patient havingesophageal cancer. In some embodiments, the evaluating step comprisesdetermining whether FGFR2:BICC1 is present in a biological sample from apatient having esophageal cancer. In some embodiments, the evaluatingstep comprises determining whether FGFR2:CASP7 is present in abiological sample from a patient having esophageal cancer. In someembodiments, the evaluating step comprises determining whether FGFR3R248C is present in a biological sample from a patient having esophagealcancer. In some embodiments, the evaluating step comprises determiningwhether FGFR3 S249C is present in a biological sample from a patienthaving esophageal cancer. In some embodiments, the evaluating stepcomprises determining whether FGFR3 G370C is present in a biologicalsample from a patient having esophageal cancer. In some embodiments, theevaluating step comprises determining whether FGFR3 Y373C is present ina biological sample from a patient having esophageal cancer. In someembodiments, the evaluating step comprises determining whether anycombination of the above FGFR mutants is present in a biological samplefrom a patient having esophageal cancer.

For patients with metastatic esophageal cancer, for example, a suitableFGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3,FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3,FGFR2:CASP7, FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof.Accordingly, in some embodiments, a patient having metastatic esophagealcancer is treated with an FGFR inhibitor if FGFR3:TACC3 v1 is present inthe sample. In some embodiments, a patient having metastatic esophagealcancer is treated with an FGFR inhibitor if FGFR3:TACC3 v3 is present inthe sample. In some embodiments, a patient having metastatic esophagealcancer is treated with an FGFR inhibitor if FGFR3:TACC3 Intron ispresent in the sample. In some embodiments, a patient having metastaticesophageal cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1 ispresent in the sample. In some embodiments, a patient having metastaticesophageal cancer is treated with an FGFR inhibitor if FGFR2:BICC1 ispresent in the sample. In some embodiments, a patient having metastaticesophageal cancer is treated with an FGFR inhibitor if FGFR2:AFF3 ispresent in the sample. In some embodiments, a patient having metastaticesophageal cancer is treated with an FGFR inhibitor if FGFR2:CASP7 ispresent in the sample. In some embodiments, a patient having metastaticesophageal cancer is treated with an FGFR inhibitor if FGFR2:CCD6 ispresent in the sample. In some embodiments, a patient having metastaticesophageal cancer is treated with an FGFR inhibitor if FGFR2:OFD1 ispresent in the sample. In some embodiments, a patient having metastaticesophageal cancer is treated with an FGFR inhibitor if any combinationof the above FGFR mutants is present in the sample.

For patients with non-small-cell lung (NSCL) adenocarcinoma, forexample, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3 v1,FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:AFF3,FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, orany combination thereof. Accordingly, in some embodiments, theevaluating step comprises determining whether FGFR3:TACC3 v1 is presentin a biological sample from a patient having NSCL adenocarcinoma. Insome embodiments, the evaluating step comprises determining whetherFGFR3:TACC3 v3 is present in a biological sample from a patient havingNSCL adenocarcinoma. In some embodiments, the evaluating step comprisesdetermining whether FGFR3:TACC3 Intron is present in a biological samplefrom a patient having NSCL adenocarcinoma. In some embodiments, theevaluating step comprises determining whether FGFR3:BAIAP2L1 is presentin a biological sample from a patient having NSCL adenocarcinoma. Insome embodiments, the evaluating step comprises determining whetherFGFR2:AFF3 is present in a biological sample from a patient having NSCLadenocarcinoma. In some embodiments, the evaluating step comprisesdetermining whether FGFR2:CASP7 is present in a biological sample from apatient having NSCL adenocarcinoma. In some embodiments, the evaluatingstep comprises determining whether FGFR3 R248C is present in abiological sample from a patient having NSCL adenocarcinoma. In someembodiments, the evaluating step comprises determining whether FGFR3S249C is present in a biological sample from a patient having NSCLadenocarcinoma. In some embodiments, the evaluating step comprisesdetermining whether FGFR3 G370C is present in a biological sample from apatient having NSCL adenocarcinoma. In some embodiments, the evaluatingstep comprises determining whether FGFR3 Y373C is present in abiological sample from a patient having NSCL adenocarcinoma. In someembodiments, the evaluating step comprises determining whether anycombination of the above FGFR mutants is present in a biological samplefrom a patient having NSCL adenocarcinoma.

For patients with non-small cell lung (NSCL) squamous cell carcinoma,for example, a suitable FGFR mutant gene panel can comprise FGFR3:TACC3v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3,FGFR2:CASP7, FGFR2:CCDC6, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, orFGFR3 Y373C, or any combination thereof. Accordingly, in someembodiments, the evaluating step comprises determining whetherFGFR3:TACC3 v1 is present in a biological sample from a patient havingNSCL squamous cell carcinoma. In some embodiments, the evaluating stepcomprises determining whether FGFR3:TACC3 v3 is present in a biologicalsample from a patient having NSCL squamous cell carcinoma. In someembodiments, the evaluating step comprises determining whetherFGFR3:BAIAP2L1 is present in a biological sample from a patient havingNSCL squamous cell carcinoma. In some embodiments, the evaluating stepcomprises determining whether FGFR2:BICC1 is present in a biologicalsample from a patient having NSCL squamous cell carcinoma. In someembodiments, the evaluating step comprises determining whetherFGFR2:AFF3 is present in a biological sample from a patient having NSCLsquamous cell carcinoma. In some embodiments, the evaluating stepcomprises determining whether FGFR2:CASP7 is present in a biologicalsample from a patient having NSCL squamous cell carcinoma. In someembodiments, the evaluating step comprises determining whetherFGFR2:CCDC6 is present in a biological sample from a patient having NSCLsquamous cell carcinoma. In some embodiments, the evaluating stepcomprises determining whether FGFR3 R248C is present in a biologicalsample from a patient having NSCL squamous cell carcinoma. In someembodiments, the evaluating step comprises determining whether FGFR3S249C is present in a biological sample from a patient having NSCLsquamous cell carcinoma. In some embodiments, the evaluating stepcomprises determining whether FGFR3 G370C is present in a biologicalsample from a patient having NSCL squamous cell carcinoma. In someembodiments, the evaluating step comprises determining whether FGFR3Y373C is present in a biological sample from a patient having NSCLsquamous cell carcinoma. In some embodiments, the evaluating stepcomprises determining whether any combination of the above FGFR mutantsis present in a biological sample from a patient having NSCL squamouscell carcinoma.

For patients with metastatic endometrial cancer, for example, a suitableFGFR mutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3,FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR2:CCDC6, orFGFR2:OFD1, or any combination thereof. Accordingly, in someembodiments, a patient having metastatic endometrial cancer is treatedwith an FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. Insome embodiments, a patient having metastatic endometrial cancer istreated with an FGFR inhibitor if FGFR3:TACC3 v3 is present in thesample. In some embodiments, a patient having metastatic endometrialcancer is treated with an FGFR inhibitor if FGFR3:TACC3 Intron ispresent in the sample. In some embodiments, a patient having metastaticendometrial cancer is treated with an FGFR inhibitor if FGFR3:BAIAP2L1is present in the sample. In some embodiments, a patient havingmetastatic endometrial cancer is treated with an FGFR inhibitor ifFGFR2:CASP7 is present in the sample. In some embodiments, a patienthaving metastatic endometrial cancer is treated with an FGFR inhibitorif FGFR2:CCDC6 is present in the sample. In some embodiments, a patienthaving metastatic endometrial cancer is treated with an FGFR inhibitorif FGFR2:OFD1 is present in the sample. In some embodiments, a patienthaving metastatic endometrial cancer is treated with an FGFR inhibitorif any combination of the above FGFR mutants is present in the sample.

For patients with breast cancer, for example, a suitable FGFR mutantgene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7,FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof. Accordingly, insome embodiments, a patient having breast cancer is treated with an FGFRinhibitor if FGFR3:TACC3 v1 is present in the sample. In someembodiments, a patient having breast cancer is treated with an FGFRinhibitor if FGFR3:TACC3 v3 is present in the sample. In someembodiments, a patient having breast cancer is treated with an FGFRinhibitor if FGFR3:TACC3 Intron is present in the sample. In someembodiments, a patient having breast cancer is treated with an FGFRinhibitor if FGFR3:BAIAP2L1 is present in the sample. In someembodiments, a patient having breast cancer is treated with an FGFRinhibitor if FGFR2:BICC1 is present in the sample. In some embodiments,a patient having breast cancer is treated with an FGFR inhibitor ifFGFR2:AFF3 is present in the sample. In some embodiments, a patienthaving breast cancer is treated with an FGFR inhibitor if FGFR2:CASP7 ispresent in the sample. In some embodiments, a patient having breastcancer is treated with an FGFR inhibitor if FGFR2:CCD6 is present in thesample. In some embodiments, a patient having breast cancer is treatedwith an FGFR inhibitor if FGFR2:OFD1 is present in the sample. In someembodiments, a patient having breast cancer is treated with an FGFRinhibitor if any combination of the above FGFR mutants is present in thesample.

For patients with hepatocellular carcinoma, for example, a suitable FGFRmutant gene panel can comprise FGFR3:TACC3 v1, FGFR3:TACC3 v3,FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3,FGFR2:CASP7, FGFR2:CCDC6, FGFR2:OFD1, FGFR3 R248C, FGFR3 S249C, FGFR3G370C, or FGFR3 Y373C, or any combination thereof. Accordingly, in someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if FGFR3:TACC3 v1 is present in the sample. In someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if FGFR3:TACC3 v3 is present in the sample. In someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if FGFR3:TACC3 Intron is present in the sample. Insome embodiments, a patient having hepatocellular carcinoma is treatedwith an FGFR inhibitor if FGFR3:BAIAP2L1 is present in the sample. Insome embodiments, a patient having hepatocellular carcinoma is treatedwith an FGFR inhibitor if FGFR2:BICC1 is present in the sample. In someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if FGFR2:AFF3 is present in the sample. In someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if FGFR2:CASP7 is present in the sample. In someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if FGFR2:CCDC6 is present in the sample. In someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if FGFR2:OFD1 is present in the sample. In someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if FGFR3 R248C is present in the sample. In someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if FGFR3 S249C is present in the sample. In someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if FGFR3 G370C is present in the sample. In someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if FGFR3 Y373C is present in the sample. In someembodiments, a patient having hepatocellular carcinoma is treated withan FGFR inhibitor if any combination of the above FGFR mutants ispresent in the sample.

Suitable pairs of primers for use in the amplifying step include thosedisclosed in Table 3. For example, in some embodiments, the FGFR mutantand pair of primers can be FGFR3:TACC3 v1 and primers having the aminoacid sequences of SEQ ID NO:5 and SEQ ID NO:6. In some embodiments, theFGFR mutant and pair of primers can be FGFR3:TACC3 v3 and primers havingthe amino acid sequences of SEQ ID NO:7 and SEQ ID NO:8. In someembodiments, the FGFR mutant and pair of primers can be FGFR3:TACC3Intron and primers having the amino acid sequences of SEQ ID NO:9 andSEQ ID NO:10. In some embodiments, the FGFR mutant and pair of primerscan be FGFR3:BAIAP2L1 and primers having the amino acid sequences of SEQID NO:11 and SEQ ID NO:12. In some embodiments, the FGFR mutant and pairof primers can be FGFR2:BICC1 and primers having the amino acidsequences of SEQ ID NO:13 and SEQ ID NO:14. In some embodiments, theFGFR mutant and pair of primers can be FGFR2:AFF3 and primers having theamino acid sequences of SEQ ID NO:15 and SEQ ID NO:16. In someembodiments, the FGFR mutant and pair of primers can be FGFR2:CASP7 andprimers having the amino acid sequences of SEQ ID NO:17 and SEQ IDNO:18. In some embodiments, the FGFR mutant and pair of primers can beFGFR2:CCDC6 and primers having the amino acid sequences of SEQ ID NO:19and SEQ ID NO:20. In some embodiments, the FGFR mutant and pair ofprimers can be FGFR2:OFD1 and primers having the amino acid sequences ofSEQ ID NO:21 and SEQ ID NO:22. In some embodiments, the FGFR mutant andpair of primers can be R248C and primers having the amino acid sequencesof SEQ ID NO:23 and SEQ ID NO:24 or SEQ ID NO:31 and SEQ ID NO:32. Insome embodiments, the FGFR mutant and pair of primers can be S249C andprimers having the amino acid sequences of SEQ ID NO:25 and SEQ ID NO:26or SEQ ID NO:33 and SEQ ID NO:34. In some embodiments, the FGFR mutantand pair of primers can be G370C and primers having the amino acidsequences of SEQ ID NO:27 and SEQ ID NO:28 or SEQ ID NO:35 and SEQ IDNO:36. In some embodiments, the FGFR mutant and pair of primers can beY373C and primers having the amino acid sequences of SEQ ID NO:29 andSEQ ID NO:30 or SEQ ID NO:37 and SEQ ID NO:38. In some embodiments, theFGFR mutant and pair of primers can be any combination of the abovedisclosed FGFR mutants and corresponding pair of primers.

The disclosed methods comprise determining whether the one or more FGFRmutants from the gene panel are present in the sample. In someembodiments, the determining step comprises sequencing the amplifiedcDNA.

In some embodiments, the method further comprises treating the patientwith an FGFR inhibitor if the one or more FGFR mutants from the genepanel are present in the sample. Suitable FGFR inhibitors for use in thetreatment methods include those previously described herein, inparticular JNJ-42756493.

Kits for Identifying the Presence of FGFR Mutant Genes

Further disclosed are kits for identifying the presence of one or moreFGFR mutant genes in a biological sample comprising: pairs of primershaving the sequences of SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12,SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ IDNO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 andSEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:25 and SEQ IDNO:26, SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, SEQID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ IDNO:36, SEQ ID NO:37, SEQ ID NO:38, or any combination thereof; andinstructions for performing an assay to detect one or more FGFR mutantgenes.

The kits can further comprise one or more probes, one or more 3′blocking oligonucleotides, or both. In some embodiments, the kits canfurther comprise one or more probes, for example, any one or more of theprobes disclosed in Table 15. In some embodiments, the kits can furthercomprise one or more 3′ blocking oligonucleotides, for example, any oneor more of the 3′ blocking oligonucleotides disclosed in Table 8. Insome embodiments, the kits can further comprise one or more probes andone or more 3′ blocking oligonucleotides. For example, in someembodiments, the kits can further comprise:

-   -   a. the pair of primers have the sequences SEQ ID NO:5 and SEQ ID        NO:6 and the probe has the sequence of SEQ ID NO:43;    -   b. the pair of primers have the sequences SEQ ID NO:7 and SEQ ID        NO:8 and the probe has the sequence of SEQ ID NO:44;    -   c. the pair of primers have the sequences SEQ ID NO:9 and SEQ ID        NO:10 and the probe has the sequence of SEQ ID NO:46;    -   d. the pair of primers have the sequences SEQ ID NO:11 and SEQ        ID NO:12 and the probe has the sequence of SEQ ID NO:47;    -   e. the pair of primers have the sequences SEQ ID NO:13 and SEQ        ID NO:14 and the probe has the sequence of SEQ ID NO:45;    -   f. the pair of primers have the sequences SEQ ID NO:15 and SEQ        ID NO:16 and the probe has the sequence of SEQ ID NO:48;    -   g. the pair of primers have the sequences SEQ ID NO:17 and SEQ        ID NO:18 and the probe has the sequence of SEQ ID NO:49;    -   h. the pair of primers have the sequences SEQ ID NO:19 and SEQ        ID NO:20 and the probe has the sequence of SEQ ID NO:50;    -   i. the pair of primers have the sequences SEQ ID NO:21 and SEQ        ID NO:22 and the probe has the sequence of SEQ ID NO:51;    -   j. the pair of primers have the sequences SEQ ID NO:23 and SEQ        ID NO:24 and the probe has the sequence of SEQ ID NO:52;    -   k. the pair of primers have the sequences SEQ ID NO:25 and SEQ        ID NO:26 and the probe has the sequence of SEQ ID NO:53;    -   l. the pair of primers have the sequences SEQ ID NO:27 and SEQ        ID NO:28 and the probe has the sequence of SEQ ID NO:54;    -   m. the pair of primers have the sequences SEQ ID NO:29 and SEQ        ID NO:30 and the probe has the sequence of SEQ ID NO:55;    -   n. the pair of primers have the sequences SEQ ID NO:31 and SEQ        ID NO:32, the probe has the sequence of SEQ ID NO:52, and the 3′        blocking oligonucleotide has the sequence of SEQ ID NO:39;    -   o. the pair of primers have the sequences SEQ ID NO:33 and SEQ        ID NO:34, the probe has the sequence of SEQ ID NO:53, and the 3′        blocking oligonucleotide has the sequence of SEQ ID NO:40;    -   p. the pair of primers have the sequences SEQ ID NO:35 and SEQ        ID NO:36, the probe has the sequence of SEQ ID NO:54, and the 3′        blocking oligonucleotide has the sequence of SEQ ID NO:41;    -   q. the pair of primers have the sequences SEQ ID NO:37 and SEQ        ID NO:38, the probe has the sequence of SEQ ID NO:55, and the 3′        blocking oligonucleotide has the sequence of SEQ ID NO:42; or    -   r. any combination thereof.

Oligonucleotide Probes

Also disclosed are oligonucleotide probes having the sequence of any oneof SEQ ID NOs:43-55. In some embodiments, the oligonucleotide probe canhave the sequence of SEQ ID NO:43. In some embodiments, theoligonucleotide probe can have the sequence of SEQ ID NO:44. In someembodiments, the oligonucleotide probe can have the sequence of SEQ IDNO:45. In some embodiments, the oligonucleotide probe can have thesequence of SEQ ID NO:46. In some embodiments, the oligonucleotide probecan have the sequence of SEQ ID NO:47. In some embodiments, theoligonucleotide probe can have the sequence of SEQ ID NO:48. In someembodiments, the oligonucleotide probe can have the sequence of SEQ IDNO:49. In some embodiments, the oligonucleotide probe can have thesequence of SEQ ID NO:50. In some embodiments, the oligonucleotide probecan have the sequence of SEQ ID NO:51. In some embodiments, theoligonucleotide probe can have the sequence of SEQ ID NO:52. In someembodiments, the oligonucleotide probe can have the sequence of SEQ IDNO:53. In some embodiments, the oligonucleotide probe can have thesequence of SEQ ID NO:54. In some embodiments, the oligonucleotide probecan have the sequence of SEQ ID NO:55.

3′ Blocking Oligonucleotide

Also disclosed herein are oligonucleotides having the sequence of anyone of SEQ ID NOs:39-42. In some embodiments, the 3′ blockingoligonucleotide can have the sequence of SEQ ID NO:39. In someembodiments, the 3′ blocking oligonucleotide can have the sequence ofSEQ ID NO:40. In some embodiments, the 3′ blocking oligonucleotide canhave the sequence of SEQ ID NO:41. In some embodiments, the 3′ blockingoligonucleotide can have the sequence of SEQ ID NO:42.

EXAMPLES Example 1 Plasmid DNA Isolation and Purification

Below is an exemplary procedure for preparing FGFR fusion plasmid DNA.

Required equipment: centrifuge, capable of 1500×g; microcentrifuge;pipettors, positive-displacement or air-displacement; vortexer; nanodropSpectrophotometer; 37° C. shaker/incubator; and an oven set to 37° C.

Required materials: frozen glycerol bacterial stock containing plasmidDNA; Kanamycin LB agar plates (Teknova #L1155); LB broth (LifeTechnologies #10855-021); Kanamycin (Sigma #K0254); plasmid purificationkit (Qiagen #12123); absolute ethanol (Sigma Aldrich #E7023);isopropanol (Sigma Aldrich #W292907); Nuclease Free Water (Non-DEPCtreated) (from IDT or Ambion #AM9932); RNase-free Barrier (Filter) Tips;RNase-free Microtube (1.5 to 2 mL VWR #10011-724); serological pipettes;and 14 ml Round bottom tubes (VWR #352057).

To recover bacteria from the glycerol stock, frozen bacteria werescraped off of the top of a glycerol stock tube using a sterile pipettip, streaked onto a LB agar plate, and placed upside down in the ovenat 37° C. overnight.

DNA plasmids were purified using Qiagen Plasmid DNA Purificationprotocol. Briefly, a single colony was picked from the streaked plateand incubated in a culture of 5 ml-LB medium containing 50 μg/mlKanamycin overnight in a 37° C. shaker at approximately 300 rpm. Thebacterial cells were harvested by centrifugation at 6000×g for 15minutes at 4° C., and the pellet was resuspended in 300 μl of buffer P1.300 μl of buffer P2 was added, mixed by inverting the tube 4-6 times,and incubated at RT (room temperature) for 5 minutes. 300 μl of chilledbuffer P3 was added, mixed immediately by inverting 4-6 times, incubatedon ice for 5 minutes, and centrifuged at maximum speed for 10 minutes.Supernatant containing plasmid DNA was removed promptly. A Qiagen-tip 20was equilibrated by applying 1 ml of buffer QBT and allowed to empty bygravity flow. The supernatant was applied to the Qiagen-tip 20 andallowed to enter the resin by gravity flow. The Qiagen-tip 20 was washedwith 2×2 ml of buffer QC and the DNA was eluted with 800 μl of buffer QFand the eluate was collected in a 1.5 ml Eppendorf tube. The DNA wasprecipitated by adding 0.7 volumes of isopropanol, mixed, andcentrifuged immediately at 15000×g for 30 minutes in a microcentrifuge.The supernatant was decanted, and the DNA pellet was washed in 1 ml of70% ethanol and centrifuged at 15000×g for 10 minutes. The supernatantwas decanted. The pellet was air-dried for 5-10 minutes and the DNA wasre-dissolved in 100 μl or suitable volume of nuclease-free water.Plasmid DNA was quantitated by Nanodrop and stored at −20° C. untilfurther use.

Example 2 Generation of NRK Cell Lines

Expression vectors expressing each of the FGFR fusions were constructed.The expression vector was then transfected into normal rat kidneyepithelial cells (NRK) cells. The stable cell lines were selected inmedia containing kanamycin following transfections. These cells werethen grown and mRNA was isolated and subjected to FGFR fusion assays toconfirm the presence of the specific FGFR fusions mRNA.

Example 3 FGFR-Fusion Cell Line Maintenance

The below protocol describes an exemplary procedure for culturing andmaintaining the NRK FGFR-fusion over-expressing cell lines. Cell linesinclude, but are not limited to: NRK/FGFR3:TACC3v1, NRK/FGFR3:TACC3 v3,NRK/FGFR3:BAIAP2L1, NRK/FGFR2: BICC1, NRK/FGFR2:CASP7, NRK/FGFR2:CCDC6,NRK/FGFR2:AFF3, NRK/FGFR2:OFD1, and NRK/EMPTY VECTOR (plasmid control).

Required equipment: biosafety cabinet, fitted with vacuum aspirationsystem; CO₂ Incubator, set to 37° C. with 5% CO₂; −80° C. freezer;liquid nitrogen tank; water bath, set to 37° C.; and a microscope.

Required materials: serological pipettes; tissue culture flasks (T75 VWR#BD353136 and/or T150 VWR #15705-074); tissue culture 0.2 μm filteringunits (Thermo Scientific #566-0020); DMEM (Dulbecco's Modified EagleMedium) cell culture media (Life Technologies, #11965-084); Fetal BovineSerum (FBS),certified, heat inactivated (Life Technologies, #10082147);PenStrep antibiotic solution (Life Technologies #15140-122);Trypsin-EDTA 0.25% solution (Life Technologies, #25200-056); DPBS(Dulbecco's Phosphate buffered solution, no calcium, no magnesium) (LifeTechnologies, #14190136); cell freezing container for cryopreservation;hand held pipetman; cell freezing media (Life Technologies, #12648-010);15 ml conical tubes (VWR #62406-2); and cryovials (VWR #89094-800).

To prepare the cell culture media, DMEM medium was prepared by combining445 ml of DMEM, 50 ml of FBS, and 5 ml of PenStrep. The prepared mediawas passed through a 0.2 μm filter unit and stored at 4° C.

To thaw frozen cells, prepared DMEM medium was warmed in the 37° C.water bath for at least 15 minutes and 15 ml of warmed medium was placedinto a T75 flask. Cells were removed from liquid nitrogen tank andplaced immediately in a 37° C. water bath until just thawed. Cryovialswere sprayed generously with 70% alcohol and the excess was wiped withpaper towels. The entire content was aliquoted into the T75 flaskcontaining DMEM. Flask was swirled gently to mix and placed in incubatorfor 24 hours. If the cells were not ready for splitting, the media waschanged to freshly prepared DMEM to remove residual freezing media. Ifcells were ready to split, each cell line was propagated once the flaskachieved 80% confluency (splitting ratio for each cell line wasdependent upon the experimental needs).

To freeze the cell lines, the cells were removed from the culture flaskand spun down in a 15 ml conical tube for 5 minutes at 1500 RPM at RT.The medium was aspirated and 6 ml of cell freezing medium was added. Thecells were mixed by pipetting up and down several times, and 1 ml ofcell solution was aliquoted into each of 5 cryovials. Cryovials withcells were placed in a cryofreezing container, which was stored in a−80° C. freezer overnight, followed by long term storage in a liquidnitrogen tank.

Example 4 FFPET SNP Assay

An exemplary workflow and protocol for performing a FFPET SNP assay isdescribed below. A similar procedure is performed for FFPET fusionassays, the results of which are shown in FIG. 2.

De-Parafinization of FFPET

Slides were subjected to increasing amounts of xylene followed byalcohol treatment in order to remove the paraffin.

FFPET RNA Extraction

The procedure for extracting RNA from breast cancer formalin fixedparaffin embedded tissue samples for downstream gene expression assay isdescribed below.

Required equipment: centrifuge with plate adapter, capable of 1500×g;microcentrifuge; pipettors, positive-displacement or air-displacement;vortexer; NanoDrop 8000; heating block capable of incubation at 37° C.,56° C. and 80° C.; and pasteur pipette (Pipet Trans EX-FT 1.5 ml pk 500,VWR #14670-329).

Required Materials: AllPrep DNA/RNA FFPE Kit (Qiagen #80234); AbsoluteEthanol (Sigma Aldrich #E7023); Isopropanol; Xylene; Nuclease Free Water(Non-DEPC treated) (from IDT or Ambion #AM9932); RNase-free Barrier(Filter) Tips; RNase-free; microtube (1.5 to 2 mL VWR #10011-724); andQiagen AllPrep DNA/RNA FFPE Kit Handbook.

RNA was extracted using the AllPrep DNA/RNA FFPE Kit. Briefly, one 1-10um section was placed in a 1.5 ml reaction tube and 800 μl of HemoDe orXylene were added. The sample was vortexed for 4 seconds 3 times,incubated for 2 minutes, vortexed for 4 seconds for 3 times andincubated for 5 minutes.

The sample was centrifuged for 2 minutes at maximum speed(12,000-14,000×g) and the supernatant was discarded by aspiration. Tubeswere capped immediately to avoid tissue from drying.

The above steps were repeated.

800 μl ethanol abs. was added, the tube was flicked to dislodge thepellet, vortexed for 4 seconds 3 times, centrifuged for 2 minutes atmaximum speed (12,000-14,000×g), and the supernatant was discarded byaspiration.

800 μl 70% ethanol was added, the tube was flicked to dislodge thepellet, vortexed for 4 seconds 3 times, centrifuged for 2 minutes atmaximum speed, and the supernatant was discarded by aspiration. Afterremoval of 70% ethanol, the tube was re-spun for 10-20 seconds and theresidual fluid was carefully removed with a fine bore pipet.

The open tubes were incubated in a heating block for 5-15 minutes at 37°C. to air dry the tissue pellet.

The pellet was resuspended by adding 150 μl Buffer PKD and the tube wasflicked to loosen the pellet. 10 μl proteinase K was added and the tubewas mixed by vortexing.

Tubes were incubated at 56° C. for 15 minutes, incubated on ice for 3minutes, and centrifuged for 15 minutes at 20,000×g.

The supernatant was carefully transferred without disturbing the pelletto a new 1.5 ml microcentrifuge tube for RNA purification. Thesupernatant was incubated at 80° C. for 15 minutes. The tube was brieflycentrifuged to remove drops from the inside of the lid. 320 μl BufferRLT was added to adjust binding conditions, and the tube was mixed byvortexing or pipetting. 1120 μl ethanol (96-100%) was added and the tubewas mixed well by vortexing or pipetting.

700 μl of the sample, including any precipitate that may have formed,was transferred to an RNeasy MinElute spin column placed in a 2 mlcollection tube, and centrifuged for 15 seconds at ≧8000×g (≧10,000rpm). The flow-through was discarded. This step was repeated until theentire sample was passed through the RNeasy MinElute spin column.

350 μl Buffer FRN was added to the RNeasy MinElute spin column andcentrifuged for 15 seconds at ≧8000×g (≧10,000 rpm). Flow-through wasdiscarded.

10 μl DNase I stock solution was added to 70 μl Buffer RDD, mixed bygently inverting the tube, and centrifuged briefly to collect residualliquid from the sides of the tube.

The DNase I incubation mix (80 μl) was added directly to the RNeasyMinElute spin column membrane, and placed on the benchtop (20-30° C.)for 15 minutes.

500 μl Buffer FRN was added to the RNeasy MinElute spin column andcentrifuged for 15 seconds at ≧8000×g (≧10,000 rpm). The flow-throughwas saved for use in the next step, as it contains small RNAs.

The RNeasy MinElute spin column was placed in a new 2 ml collection tube(supplied). The flow-through from the previous step was applied to thespin column and centrifuged for 15 seconds at ≧8000×g (≧10,000 rpm).Flow-through was discarded.

500 μl Buffer RPE was added to the RNeasy MinElute spin column andcentrifuged for 15 second at ≧8000×g (≧10,000 rpm) to wash the spincolumn membrane. Flow-through was discarded.

500 μl Buffer RPE was added to the RNeasy MinElute spin column andcentrifuged for 15 seconds at ≧8000×g (≧10,000 rpm) to wash the spincolumn membrane. Collection tube with the flow-through was discarded.

The RNeasy MinElute spin column was placed in a new 2 ml collection tubeand centrifuged at full speed for 5 minutes. The collection tube withthe flow-through was discarded.

The RNeasy MinElute spin column was placed in a new 1.5 ml collectiontube, 30 μl RNase-free water was added directly to the spin columnmembrane, incubated for 1 minute at room temperature, and centrifuged atfull speed for 1 minute to elute the RNA.

The RNA samples were immediately stored in −80° C. freezer.

cDNA Synthesis

Disclosed below is a procedure of cDNA synthesis for the FFPET SNPAssays using Real time PCR (RT-PCR) analysis.

Required equipment: centrifuge with plate adapter, capable of 1500×g,microcentrifuge; pipettors (preferred single and multi-channelpipettor), positive-displacement or air-displacement; vortexer; andGeneAmp® PCR System 9700 (ABI #4314879) or equivalent.

Required materials: High Capacity cDNA Reverse Transcriptase Kit withRNase Inhibitor, 200 reactions (ABI #4374966); Nuclease Free Water(Non-DEPC treated) (from IDT) or equivalent; RNase-free Barrier (Filter)Tips; RNase-free Microtube (1.5 to 2 mL VWR #10011-724); MicroAmp™Optical 96-Well Reaction Plates (Life Technologies, #4306736); andsealing film (VWR #60941-072).

Following the RNA extraction (disclosed above) RNA sample tube(s) werekept on ice.

The kit components were used to prepare 2× Reverse Transcription (RT)Master Mix for all reactions, including 1 negative (water) control.Components were thawed on ice for approximately 15 minutes, gentlyinverted to mix and centrifuged briefly to bring down the solution. Allreagents were returned to the ice. Tubes were not vortexed.

One Master Mix was prepared on ice in a 1.5 ml tube for the appropriatenumber of reactions (#reactions+10%, per 20-μL reaction) by combiningthe following amount of reagent per one reaction: 2 μl 10× RT BufferMix; 0.8 μl 25× dNTP Mix; 2 μl 10× RT Random Primers; 1 μl 50 U/μLMultiScribe Reverse Transcriptase; 1 μl RNase inhibitor; and 3.2 μlNuclease/RNase free H₂O.

The Master Mix was vortexed several times (5 to 10) to mix andcentrifuged briefly (1500×g, 5 to 10 seconds). 10 μl of the reaction mixwas added to the appropriate wells of a 96-well plate.

The RNA samples were diluted to a concentration of 20 ng/μl. 10 μL ofeach RNA sample was added, including the water negative control, to theappropriate corresponding wells of the 96-well plate to a final reactionvolume of 20 μL. The wells were mixed gently by pipetting up and down 3times, sealed with a plate seal, and centrifuged briefly (1500×g for 60seconds). Plates were kept on ice until ready to load in thermocycler.

The reaction plate was loaded into ABI 9700 Thermal Cycler in Clean Labor Workstation and run using the following reverse-transcription programwith a reaction volume of 20 82 l:

-   -   Step 1: 25° C. for 10 minutes    -   Step 2: 37° C. for 120 minutes    -   Step 3: 85° C. for 5 seconds    -   Step 4: 4° C. infinite hold

Synthesized cDNA was stored at −20° C. for next step ofPre-amplification.

Preamplification Assay Pool Mixture Preparation

The preamp assay pool mixture associated with the FFPET SNP AssayPre-amplification Protocol was prepared as described below.

Required equipment: microcentrifuge; pipettors, positive-displacement orair-displacement; and vortexer.

Required materials: Nuclease Free Water (Non-DEPC treated) (from IDT) orequivalent; IDTE pH 8.0 (1× TE Solution) (IDT Technologies); RNase-freeBarrier (Filter) Tips; and RNase-free Tubes (1.5 to 2 mL VWR#10011-724).

All TaqMan SNP Assays are ordered from Applied Biosystems, LifeTechnologies, Inc.

100 μL of 20× SNP assays were prepared.

To prepare 0.2× Preamp Assay Pool, all assays were thawed on ice forapproximately 15 minutes. The following volume of components was addedto a 1.5 ml tube:

TABLE 4 Stock Volume Needed for 200 ul Target Concentration Preamp Stock(ul) Preamp Stock 1 FGFR3 S249C 20X 2 IDTE 198 Total Volume 200 PreampStock 2 FGFR3 R248C 20X 2 IDTE 198 Total Volume 200 Preamp Stock 3 FGFR3Y373C 20X 2 IDTE 198 Total Volume 200 Note: The above volumes are forthe preparation of 200 μl of 0.2X preamp assay pool. Volumes can beadjusted accordingly depending on the number of samples being tested.

The 0.2× PreAmp Assay Pool was vortexed briefly to mix (5 to 10 seconds)and centrifuged briefly (1500×g, 5-10 seconds). 100 μL of PreAmp PrimerPool was aliquoted into 1.5 ml tubes and stored at −20° C.

Pre-Amplification for the Breast Cancer Formalin-Fixed Paraffin EmbeddedTissue SNP Assay Using Real Time PCR (RT-PCR) Analysis

Required equipment: centrifuge with plate adapter, capable of 1500×g;microcentrifuge; pipettors, positive-displacement or air-displacement;vortexer; GeneAmp® PCR System 9700 (ABI #4314879) or equivalent.

Required Materials: TaqMan® PreAmp Master Mix (2×) (Life Technologies#4391128); 0.2× Pooled Assay Mix (see Assay Preparation and HandlingProtocol); 1× IDTE Buffer (10 mM Tris/0.1 mM EDTA, pH7.5, from IDT) orequivalent; Nuclease Free Water (Non-DEPC treated) (from IDT) orequivalent; RNase-free Barrier (Filter) Tips; RNase-free Microtube (1.5to 2 mL VWR #10011-724); MicroAmp™ Optical 96-Well Reaction Plates (LifeTechnologies, #4306736); MicroAmp® Optical Adhesive Film (AppliedBiosystems PN 4311971); deep well plates (VWR #47734-788); foil seals(VWR #60941-126).

Samples were prepared by placing the cDNA and 0.2× assay mix pool on iceto thaw, approximately 5 minutes, and centrifuging the plate briefly(1500×g for 5 to 10 seconds).

The kit components were used to prepare 2× PreAmp Master Mix. The kitcomponents were allowed to thaw on ice for approximately 5 minutes.After all reagents were thawed, the tubes were gently inverted to mixand briefly centrifuged to bring down the solution. All reagents werereturned to the ice. The tubes were not vortexed.

In a Clean Lab or Biosafety hood, each Master Mix was prepared for theappropriate number of reactions on ice by combining the required volumesof reagents as indicated Table 5 below (#reactions+10%):

TABLE 5 Volume (μL) for One Component Reaction Master Mix 1 2X TaqManPreAmp Master Mix 12.5 0.2X Assay Pool 1 6.25 Total volume 18.75 MasterMix 2 2X TaqMan PreAmp Master Mix 12.5 0.2X Assay Pool 2 6.25 Totalvolume 18.75 Master Mix 3 2X TaqMan PreAmp Master Mix 12.5 0.2X AssayPool 3 6.25 Total volume 18.75 Assay pools contain primers and probes.

To prevent cross-priming of SNP assays, all 5 assays were split into 3preamp reaction per sample.

Each Master Mix was vortexed several times (5 to 10) to mix, followed bya brief centrifuge (1500×g, 5 to 10 seconds). 18.75 μL of each MasterMix was aliquoted to the appropriate wells in a 96-well reaction plate.6.25 μL of each cDNA samples, including water negative control well, wastransferred into the appropriate wells in the Master Mix reaction platefor each preamp reaction. The sample was mixed gently by pipetting upand down 3 times and the cap was closed. The plate was brieflycentrifuged (1500×g for 60 seconds) and kept on ice until ready to loadin thermocycler.

The reaction plate ABI 9700 Thermal Cycler was loaded and run using thefollowing program:

-   -   Step 1: 95° C. for 10 minutes    -   Step 2: 95° C. for 15 seconds    -   Step 3: 60° C. for 4 minutes    -   Step 4: Set Step 2-3 for 10 cycles

If a Gold or Silver Block was Used, Max Mode was Selected and Ramp Ratewas set at 77%.

If an Aluminum Block was Used, Standard Mode (No Rate Change) wasSelected.

-   -   Step 5: 4° C. infinite hold    -   Reaction volume set to 25 μL

The PreAmp reaction plate was centrifuged briefly (1500×g for 60seconds) after PreAmp completion. 100 μl of IDTE was added to theappropriate wells of a new deep 96-well plate and 25 μL of each PreAmpproduct was transferred to the corresponding wells to have finaldilution volume of 125 μL. The each well was mixed by pipetting up anddown 3 times, the plate was sealed with foil adhesive, the plate wascentrifuged briefly (1500×g for 5 to 10 seconds), and the PreAmp productwas stored at −20° C. until further use.

FFPET SNP Assay—Real Time PCR

Disclosed below is the procedure for the Formalin-Fixed ParaffinEmbedded Tissue SNP Assay using Real time PCR analysis.

Required equipment: centrifuge with plate adapter, capable of 1500×g;microcentrifuge; pipettors (preferred single and multi-channelpipettor), positive-displacement or air-displacement; vortexer; and ABIViiA 7 real time PCR instrument (Life Technologies).

Required materials: TaqMan Genotyping Master Mix (Life Technologies#4371355); SNP Assays; Nuclease Free Water (Non-DEPC treated, from IDT)or equivalent; RNase-free Barrier (Filter) Tips; RNase-free Microtube(1.5 to 2 mL VWR #10011-724); MicroAmp® Optical Adhesive Film (AppliedBiosystems PN 4311971); and MicroAmp™ Optical 384-Well Reaction Plates.

Table 15 lists the sequences of the probes used during the Real Time PCRassays.

To prepare the samples, in a Clean Lab or Workstation, SNP assays wereplaced on ice to thaw for approximately 5 minutes. All reagentsprotected from light, to protect exposure of the fluorescent probes.Diluted PreAmp plates were placed on ice to thaw in a Dirty Lab orWorkstation after preparing Genotyping Master Mix.

To prepare genotyping master mix, the Genotyping Master Mix was thawedon ice for approximately 5 minutes. The Master Mix (MM) was prepared inthe required number of tubes on ice. The required volumes of reagentswere combined in the appropriate labeled tubes as indicated in Table 6below (# reactions+10%):

TABLE 6 Volume (μL) Component for One Reaction 2X Genotyping Master Mix10 20X SNP Assay 1 RNase-free Water 4 Total volume 15 20X SNP assay mixcontains primers, probes, and blocking oligos.

The Master Mix was vortexed several times (5 to 10) to mix and thencentrifuged briefly (1500μg, 5 to 10 seconds). 15 μl of each Master Mixwas added to the appropriate wells of a MicroAmp™ Optical 384-WellReaction Plates. The reaction plates were sealed with optical adhesivefilm.

The plate with 1:5 diluted PreAmp product was placed on ice forapproximately 5-10 minutes to thaw. Using a multi-channel pipettor, 5 82L of each diluted PreAmp product was transferred to the appropriatecorresponding wells. The reaction plate was sealed with optical adhesivefilm and centrifuged briefly (1500×g for 60 seconds). Plates were kepton ice until ready to load in thermocycler.

The following conditions were run using the viiA 7 Software with thevolume set at 20 μl:

TABLE 7 Stage Repetitions Process Temperature Time 1 1 Initial 60° C.0.5 minutes 2 1 DNApol Activation 95° C. 10 minutes 3 40 Denature 95° C.15 seconds Anneal/Extend 60° C. 1 minutes 4 1 Post-Read 60° C. 30secondsFGFR SNP-Specific qRT-PCR

The detection of rare somatic mutations in an excess of wild typealleles is increasingly important in cancer diagnosis. When themutations of interest are close to each other, detection becomeschallenging. To aid in the identification of FGFR SNPs from FFPET, aSNP-specific qRT-PCR assay was developed, in which SNP-specificamplification using Taqman MGB probes combined with the 3′ dideoxy wildtype (WT) allele blocker was used. The assay prevented non-specificbinding, improved the number of on-target amplification, minimized thefalse positive signals from the WT alleles, and increased thesensitivity of the assay. This RNA based SNP detection assay, combinedwith the pre-amplification step in the assay, boosts the low or the raremutant signals.

An exemplary strategy for SNP-specific qRT-PCR using a 3′ dideoxy WTblocker oligonucleotide is shown in FIG. 3, and an exemplary FFPE samplevalidation strategy is illustrated in FIG. 4. Briefly, qRT-PCR wasperformed using the FGFR SNP primers in the presence of a 3′ dideoxy WTblocker oligonucleotide, which was complementary to, and contained ashort stretch of nucleotides flanking, the WT allele. Binding of theblocker oligonucleotide to the WT allele prevented applification of theWT allele, while the FGFR SNP primers bound to and specificallyamplified the FGFR SNP. The 3′ dideoxy WT blocker oligonucleotides usedin the FGFR SNP-specific qRT-PCR are shown in Table 8. The FGFR SNPprimers used in the FGFR SNP-specific qRT-PCR were: SEQ ID NO:31 and SEQID NO:32 (FGFR3 R248C); SEQ ID NO:33 and SEQ ID NO:34 (FGFR3 S249C); SEQID NO:35 and SEQ ID NO:36 (FGFR3 G370C); and SEQ ID NO:37 and SEQ IDNO:38 (FGFR3 Y373). Table 15 lists the sequences of the probes usedduring the real time PCR assays.

TABLE 8 3′ dideoxy WT Target blocker oligonucleotide FGFR3 R248CTGGAGCGCTCCCCGCA-ddC (SEQ ID NO: 39) FGFR3 S249C GACGTGCTGGAGRGCTC-ddC(SEQ ID NO: 40)* FGFR3 G370C CTGACGAGGCGGGCAG-ddC (SEQ ID NO: 41)FGFR3 Y373 GTGTGTATGCAGGCATCCTCAG-ddC (SEQ ID NO: 42) *R can be A or G.3′ WT blocking oligo will have 50% A and 50% G at that particularposition during the synthesis (purified by manufacturer to provide A orG at that particular position).

Samples for validatation studies were prepared as shown in Table 9.Exemplary validation data of the SNP-specific qRT-PCR using a 3′ dideoxyWT blocker oligonucleotide for FGFR3 G370C, FGFR3 Y373, FGFR3 S249C, andFGFR3 R248C is illustrated in FIGS. 5A-5D, respectively. Raw Ct (cyclethreshold) data for the FFPE samples with SNP-specific qRT-PCR with 3′dideoxy WT blocker oligonucleotides are shown in Table 10. The dataderived from DNA and RNA using different platforms/techniques suggeststhat SNP-specific PCR with the 3′ blocking nucleotide is a robust,reliable and a sensitive assay. The validation data suggests that onemutant allele/SNP can be detected in a large excess of WT-bearinggenomic DNA, thus emphasizing the sensitivity and the specificity ofeach assay.

TABLE 9 Sample % Mutant 1 100 2 20 3 4 4 0.8 5 0 (100% WT) RNA fromStable cell lines expressing each FGFR3 SNPs (R248C, S249C, G370C,Y373C) and FGFR3 WT

TABLE 10 FGFR3 SNPs - SNP-Specific PCR with Janssen Dideoxy WT Blocker(Ct *) R&D Pt Id# R248C S249C G370C Y373C FMI/NGS ver1.0 7502 >3528.03 >35 >35 S249C S249C 10000305 >35 >35 >35 >35 WT WT 33000127 >3520.92 >35 >35 S249C S249C 33000118 >35 29.35 >35 >35 S249C S249C10000306 >35 >35 >35 24.30 Y373C Y373C 34000226 >35 >35 >35 >35 WT WT16446 >35 28.03 >35 >35 S249C S249C * Mean of two Cts FMI/NGS = Nextgeneration Sequencing technique wherein DNA is used as an template toidentify the mutations (without 3′ blocking oligonucleotide); JanssenR&D = performed on RNA template (without the 3′ blockingoligonucleotide); SNP-specific PCR performed on RNA template with the 3′blocking nucleotide.

Example 5 Validation of Custom FGFR Fusion Gene Detection AssayGeneration of Positive Controls for FGFR Fusion Assays

FGFR fusion “synthetic mini-genes,” plasmids encoding FGFR fusions, andstable cell lines containing FGFR fusions were generated. Briefly,Synthetic mini genes were artificially constructed by linking a seriesof nucleotides, of about 100 base pairs, to each other corresponding tothe target DNA sequence of the gene of interest. Plasmids encoding FGFRfusions were generated by cloning cDNA encoding the various FGFR fusiongenes into an expression vector. Stable cell lines containing FGFRfusions were generated by transfecting plasmids encoding FGFR genes intonormal rat kidney epithelial cells (NRK cells). The stable cell lineswere selected under the G418 antibiotic. The FGFR fusion Taqman assaywas performed on the total RNA isolated from these cell lines to confirmthe successful generation of stable cell line(s) expressing the FGFRfusion(s). The stable cell lines expressing FGFR fusions are used apositive control. Table 15 lists the sequences of the probes used duringthe real time PCR assays.

Analysis of Lower Limit of Quantitation and Efficiency of FGFR FusionAssays

To determine the lower limit of quantitation (LLOQ) and efficiency ofthe FGFR fusion gene assays, FGFR fusion products were generated byTaqMan PCR (as described in Example 4) and confirmed by SangerSequencing (FIG. 2). 100 pg of fusion positive DNA was mixed with normalhuman cDNA (confirmed fusion-negative), serially diluted 1:10, andanalyzed using the Applied Biosystems ViiA7 Software v1.1. Efficiencystandard curves are shown in FIG. 6. FGFR fusion LLOQ and efficiency areshown in Table 11.

TABLE 11 Assay LLOQ Efficiency FGFR3:TACC3 V1 1.0 fgm 104% FGFR3:TACC3V3 10.0 fgm  104% FGFR3:TACC3 Intron 0.1 fgm 103% FGFR3:BAIAP2L1 1.0 fgm101% FGFR2:AFF3 0.1 fgm 106% FGFR2:BICC1 10.0 fgm  105% FGFR2:CASP7 0.1fgm 109% FGFR2:CCDC6 1.0 fgm 106% FGFR2:OFD1 0.1 fgm 96.6% 

The FGFR fusion gene assay was next validated in fusion gene-positivecell lines. FGFR fusion gene expression, serial dilutions were preparedby spiking fusion protein-positive cells lines into a fusionprotein-negative cell line. For example, a 1:2 serial dilution wasprepared for both FGFR3:TACC3v1 and FGFR3:BAIAP2L1 and spiked into 1million BAF cells. RNA was isolated (using Qiagen Rneasy kit), followedby RT-PCR, preamplification of cDNA, and TaqMan Real Time PCR for thetargeted FGFR fusion gene. As shown in Table 12, both the FGFR3:TACC3v1and FGFR3:BAIAP2L1 Fusion Gene TaqMan assays are able to detect thefusion target in 31 out of 1 million fusion-negative cells (sensitivityof 0.003%).

TABLE 12 Percent SW780 of Fusion-Positive RT112 FGFR3:BAIAP2L1FGFR-fusion Cells vs FGFR3:TACC3v1 Average Ct Cell Count BackgroundAverage Ct (n = 2) (n = 2) Positive 1.00E+06   100% 17.56 20.35 Control1000 0.1000% 27.95 28.61 500 0.0500% 29.11 28.91 250 0.0250% 29.62 30.14125 0.0125% 30.26 31.43 62.5 0.0063% 31.19 31.69 LLOD 31.25 0.0031%32.59 32.97 15.6 0.0016% 34.91 >40 0 0.0000% 0.00 >40 RT112 and SW780 =commercially available bladder cancer cell lines harboring the FGFRfusions (from American Type Culture Collection).

Example 6 Validation of Custom FGFR SNP Detection Assay Evaluation ofFGFR3 Mutations in Bladder Cancer

The R248C, S249C, and Y373C SNPs were observed in approximately 8%,approximately 61%, and approximately 19% of bladder cancer samplestested, respectively.

Example 7 Analysis of Cancer Samples

Samples were analyzed using the same procedure as described in example4. The results are shown in Table 13 and FIG. 7. Table 13 shows the FGFRfusion prevalence in different cancers. FGFR fusions detected in FFPEsamples from different cancers such as bladder (primary and metastatic),NSCLC (adenocarcinoma and squamous), ovarian, esophageal (primary andmetastatic), head and neck (H&N; primary and metastatic), endometrial(metastatic), breast, and prostate cancer using the qRT-PCR method. AllFGFR fusions tested were negative for prostate cancer samples.FGFR3:TACC3intron fusion was negative in bladder (primary), NSCLC(squamous), ovarian and esophageal (primary), H&N (primary andmetastatic) and breast. FGFR2:OFD1 fusion was negative in bladder(primary and metastatic), NSCLC (adenocarcinoma), ovarian and esophageal(primary and metastatic). FGFR2:CCDC6 fusion was negative in bladder(primary and metastatic), NSCLC (adenocarcinoma), ovarian and esophageal(primary) and H&N (primary and metastatic)

FIG. 8 is an exemplary representation of FGFR fusion gene and mutationstatus in NSCLC adenocarcinoma and squamous cell carcinoma. In FGFRfusion positive NSCLC adenocarcinoma samples, 3/17 samples were positivefor EGFR mutation, 3/17 samples were positive for KRAS mutation, and1/17 samples were positive for cMET mutation. No EGFR, KRAS, or cMETmutations, however, were observed in FGFR fusion positive NSCLC squamouscell carcinoma samples.

TABLE 13 NSCLC Bladder Bladder NSCLC Squa- Ovar- Eso Eso H&N H&N EndoPros- primary Mets Adeno mous ian Primary Mets Primary Mets Mets Breasttate (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) FGFR3: 1/22 5/483/89 2/125 4/94 2/41 2/42 1/37 0/40 5/46 3/112 0/72 TACC3v1 (4.55)(10.47)  (3.37) (1.60) (4.26) (4.88) (4.76) (2.70) (0.00) (10.87) (2.69) (0.00) FGFR3: 1/22 2/48 9/89 5/129 5/94 1/41 10/42  0/37 0/402/46 6/112 0/72 TACC3v3 (4.55) (4.20) (13.90)  (3.38) (5.32) (2.44)(23.81)  (0.00) (0.00) (4.35) (5.36) (0.00) FGFR3: 0/22 0/48 3/89 0/1250/94 0/41 1/42 0/37 0/40 2/46 0/112 0/72 TACC3In- (0.00) (0.00) (3.37)(0.00) (0.00) (0.00) (2.38) (0.00) (0.00) (4.35) (0.00) (0.00) tronFGFR3: 2/17 19/44  5/89 3/115 1/94 0/41 25/42  2/37 34/40  22/46 56/112  0/72 BAIAP2L1 (11.77)  (43.18)  (5.62) (2.61) (1.06) (0.00)(59.52)  (5.41) (85.00)  (47.83)  (50.00)  (0.00) FGFR2: 1/22 4/48 0/892/123 8/94 2/41 1/42 0/37 0/40 0/46 3/112 0/72 BICC1 (4.55) (8.33)(0.00) (1.63) (8.51) (4.88) (2.40) (0.00) (0.00) (0.00) (2.70) (0.00)FGFR2: 1/17 19/44  1/89 2/111 2/94 0/41 8/42 0/37 0/40 0/46 10/112  0/72AFF3 (5.88) (43.18)  (1.12) (1.80) (2.31) (0.00) (19.05)  (0.00) (0.00)(0.00) (8.90) (0.00) FGFR2: 7/16 20/45  1/89 6/114 24/94  2/41 1/42 4/373/40 8/46 12/112  0/72 CASP7 (43.75)  (44.44)  (1.12) (5.26) (25.53) (4.88) (2.40) (10.81)  (7.50) (17.40)  (10.70)  (0.00) FGFR2: 0/22 0/480/89 6/109 0/94 0/41 4/42 0/37 0/40 6/46 3/112 0/72 CCDC6 (0.00) (0.00)(0.00) (5.50) (0.00) (0.00) (9.52) (0.00) (0.00) (13.04)  (2.70) (0.00)FGFR2: 0/17 0/44 0/89 1/121 0/94 0/41 1/42 0/37 3/40 3/46 10/112  0/72OFD1 (0.00) (0.00) (0.00) (0.83) (0.00) (0.00) (2.40) (0.00) (7.50)(6.52) (8.90) (0.00) Eso = Esophageal; Endo = Endometerial

Example 8 Treatment of Patients with Advanced Solid Tumors

A clinical trial was conducted in which patients having various solidtumors expressing the FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR2:CCDC6 andFGFR2:BICC1 fusion genes were treated with JNJ-42756493. FIG. 9illustrates exemplary results from phase I patient samples, in whichFGFR fusions in Phase I JNJ-427493 (EDI10001) trial samples weredetected using the qRT-PCR assay. All FGFR fusion assays were runsimultaneously with positive controls (ST) and GAPDH for quality controlassessment of the RNA. A) Graphical representation of the qRT-PCR datagenerated for pt #1000081: positive only for FGFR2:BICC1 fusion (insetshows details of the Ct values for FGFR2:BICC1 fusion, ST-positivecontrol and GAPDH). B) Graphical representation of the qRT-PCR datagenerated for pt #33000158: positive only for FGFR3:TACC3v1 fusion(inset shows details of the Ct values for FGFR3:TACC3v1 fusion,ST-positive control and GAPDH). C) Graphical representation of theqRT-PCR data generated for pt #34000123: positive only for FGFR2:CCDC6fusion (inset shows details of the Ct values for FGFR2:CCDC6 fusion,ST-positive control and GAPDH). D) Graphical representation of theqRT-PCR data generated for pt #340000115: positive for FGFR3:TACC3v1,FGFR3:TACC #v3 and FGFR2:CCDC6 fusions (inset shows details of the Ctvalues for FGFR fusions, ST-positive controls and GAPDH).

FIG. 10 represents an exemplary Phase I Study design for aFirst-In-Human Study of JNJ-42756493 in patients with advanced solidtumor. Shown is a graphical depiction of a traditional 3+3 design doseescalation method for the phase I clinical trial. The dose escalationphase aimed to establish the maximum tolerated dose (MTD) andrecommended Phase II dose (RPD). The Part 1 arm was used to determinethe intermittent dosing schedule, i.e., 7 days on and seven days off (10mg/kg and 12 mg/kg). The Part 2 arm was used to determine the PDbiomarkers (pharmacodynamics biomarkers; makers examined to link theeffect of the drug to the target and biological tumor response) whereinthe biopsy and blood sample were tested. The Part 3 arm was used thedose expansion cohort and included accrual of additional patients inspecific indications (NSCLC,SCLC, breast and solid tumors) withdifferent eligibility criteria (FGFR aberrations:translocation/mutation/amplifications) to further characterize thetoxicity profiles of the JNJ493.

Evaluation of Clinical Activity

Significant clinical responses (RECIST) were observed at 9 mg dosingonce a day (QD), 12 mg QD and 12 mg 7 d on/off in patients with the FGFRfusion genes. (FIG. 11; represents all dosing regimens).

Example 9 Generation of FGFR Fusion Stably Transfected RK3E Cells FGFRFusion Overexpressing Cell Lines

RK3E (rat kidney epithelial cells) cells were purchased from ATCC(Manassas, Va., USA) and cultured in DMEM supplemented with FBS andantibiotics (Invitrogen, Grand Island, N.Y., USA). FGFR fusion geneconstructs were designed and cloned into the pReceiver expression vector(Genecopoeia, Rockville, Md., USA), which contains an HA-tag. Cloneswere transfected into RK3E cells using the Amaxa Cell Line Nucleofector(Lonza, Basel, Switzerland) following the manufacturer's protocol. Thestably transfected cells were selected in complete medium with 800 ug/mlof G418 (Invitrogen). Overexpression of the fusions in the stablytransfected cells was confirmed by real-time PCR and immunoblottingusing an anti-pFGFR antibody (FIG. 12). As shown in FIG. 12, the stablecell lines showed expression of active FGFR fusion kinases, as exhibitedby the expression of phosphorylation of FGFR.

Colony Formation Assay

Anchorage-independent growth of the FGFR fusion stably transfected RK3Ecells was tested. 1 ml culture medium with 0.8% low melting pointagarose was first plated into each of three wells of a six-well plate.After the agar solidified, each well received another 1 ml of 0.4% agarin culture medium containing 100 cells. After 14 days, colonies werefixed and stained with 0.1% cresyl crystal violet. The number ofcolonies was determined microscopically by manual counting fromtriplicate wells for each cell line. A representative view of eachfusion-overexpressing cell line is shown in FIG. 13A.Anchorage-independent cell growth in soft-agar could be detected in theFGFR fusion stably transfected cells, but not in the empty vectorcontrol. FIG. 13B represents a quantitative analysis of colonies in softagar for the FGFR fusion stably transfected RK3E cells and empty vectorcontrol. All experiments were carried out in duplicate and the resultsare expressed as colonies/100 cells plated. All of the FGFR fusionstested induced anchorage independent growth, highlighting theirtransforming ability

Downstream Target Expression

FGFR fusion stably transfected RK3E cells were plated in complete growthmedium, serum starved overnight, then re-fed with 0.5% FBS growth media.Cells were treated with 1 μM of JNJ-42756493, AZD4547 or NVP-BGJ398 inthe presence of ligands for 1 hour. For immunoblotting, whole celllysates were collected in RIPA buffer (Thermo Scientific, Waltham,Mass., USA) and sample protein concentration was assayed using BCAProtein Assay (Thermo Scientific). Equal amounts of protein (30 pg perlane) were loaded onto on 4-12% Bis-Tris gels (Invitrogen) before anSDS-page was performed. Proteins were transferred to nitrocellulosemembranes and probed with antibodies against p-FGFR, total-FGFR2,p-MAPK, total-MAPK, p-S6, total S6, B-actin (Cell Signaling Technology,Danvers, Mass., USA), and total-FGFR3 (Santa Cruz, Dallas, Tex., USA).The membranes were blocked with Odyssey blocking buffer for 1 h at roomtemperature and incubated overnight at 4° C. in a primary antibodysolution diluted in Odyssey blocking buffer (1:1000). After three washesin 0.1% Tween tris buffered saline (TBST), the membranes were probedwith goat anti-mouse or donkey anti-rabbit IR-Dye 670 or 800 cw labeledsecondary antisera in Odyssey blocking buffer for 1 h at roomtemperature. Washes were repeated after secondary labeling and themembranes were imaged using a LiCor Odyssey scanner and the Odyssey 3.0analytical software (LiCor, Lincoln, Nebr., USA). Effects ofJNJ-42756493 was compared with AZD4547 and NVP-BGJ398. As shown in FIGS.14A-14H, treatment with JNJ-42756493, AZD4547 and NVP-BGJ398 (lanes 2-4in each blot) inhibited phosphorylation of FGFR and downstream targetsi.e. MAPK and S6.

Drug Response Testing for FGFR fusion Overexpressing Cell Lines

FGFR fusion stably transfected RK3E cells were seeded into 96 wellplates (1000 cells/well) in triplicates in complete growth medium plusand the ligands FGF-1 and FGF-2. After 24 hours, cells were serumstarved overnight, then re-fed with 0.5% FBS growth media. 72 hoursafter plating, cells were treated with various concentrations of an 18point 1:3 dilution series, starting at 10 μM, of JNJ493, AZD4547 (AZD),and NVP-BGJ398 (NVS). The Microtiter plates were then incubated for 72hours and assayed for adenosine triphosphate (ATP; a marker ofmetabolically active cells) content using the Cell Titer-Glo®Luminescent Cell Viability assay (Promega Corp., Madison, Wis., USA)following the manufacturer's instructions, with modifications. Briefly,cells were allowed to equilibrate to room temperature, at which time a1:1 mixture of Cell Titer-Glo® reagent was added. Cells were then placedon an orbital shaker for 2 minutes and incubated for 10 minutes at roomtemperature to stabilize the luminescent signal. The luminescence wasquantified and measurements were conducted using an Envision Multilabelplate reader (Perkin Elmer; Waltham, Mass., USA). IC₅₀ values (shown inTable 14) were calculated using GraphPad Prism 5.0. As shown in Table14, cells harboring the FGFR fusions showed sensitivity to the FGFRinhibitor JNJ-42756493, AZD4547 and NVP-BGJ398 in vitro, withJNJ-42756493 exhibiting enhanced sensitity (nanomolar concentrationrange) when compared to AZD4547 and NVP-BGJ398, whereas the empty vectorcontrol did not.

TABLE 14 Stimulated Proliferation (IC50) RK3E-Transgene JNJ493 (nM) AZD(nM) NVS (nM) Vector 7010 8011 >10 μM AFF3 0.1133 2.809 2.273 BAIA2PL10.3211 11.54 5.162 BICC1 0.3303 6.448 18.19 CASP7 0.4718 4.107 241.5CCDC6 0.1894 13.36 10.72 OFD1 0.2303 7.259 15.99 TACC3-V1 0.2915 16.532.594 TACC3-V3 0.2706 8.664 4.092 FGFR2 >10 μM 6501 >10 μM FGFR3 >10 μM5686 6344 KRAS 1621 1478 2136 AZD = AZD4547; NVS = NVP-BGJ398

TABLE 15 Target Probe Sequences FGFR3TACC3 V1 TCCACCGACGTAAAGG(SEQ ID NO: 43) FGFR3TACC3 V3 TCCACCGACGTGCCAG (SEQ ID NO: 44)FGFR2BICC1 CCAATGAGATCATGGAGG (SEQ ID NO: 45) FGFR3TACC3 IntronCCTTCTGGCCCAGGTG (SEQ ID NO: 46) FGFR3BAIAP2L1 CACCGACAATGTTATGG(SEQ ID NO: 47) FGFR2AFF3 TCACAACCAATGAGGAGAGT (SEQ ID NO: 48)FGFR2CASP7 CTGCCATCTCATTGGT (SEQ ID NO: 49) FGFR2CCDC6 AATGAGCAAGCCAGGGC(SEQ ID NO: 50) FGFR2OFD1 AAGTTGTGTCTCATTGGTT (SEQ ID NO: 51)FGFR3 R248C CTGGAGTGCTCCCC (SEQ ID NO: 52) FGFR3 S249C AGCGCTGCCCGCA(SEQ ID NO: 53) FGFR3 G370C GCGTGCAGTGTGTAT (SEQ ID NO: 54) FGFR3 Y373CTGCACACACACTGC (SEQ ID NO: 55)

Those skilled in the art will appreciate that numerous changes andmodifications can be made to the preferred embodiments of the inventionand that such changes and modifications can be made without departingfrom the spirit of the invention. It is, therefore, intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

The disclosures of each patent, patent application, and publicationcited or described in this document are hereby incorporated herein byreference, in its entirety.

Nucleotide Sequence of FGFR Fusion Genes

The nucleotide sequences for the FGFR fusion cDNA that were engineeredinto expression vectors is provided in Table 16. The underlinedsequences correspond to either FGFR3 or FGFR2, the sequences in normalfont represent the fusion partners and the sequence in italic fontsrepresent the intron sequence of the FGFR3 gene.

TABLE 16 FGFR3:TACC3 v1 >ATGGGCGCCCCTGCCTGCGCCCTCGCGCT (3271 base pairs)CTGCGTGGCCGTGGCCATCGTGGCCGGCGC (SEQ ID NO: 56)CTCCTCGGAGTCCTTGGGGACGGAGCAGCG CGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGAGCAGTTGGT CTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGG GCCCACTGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGG GCCCCAGCGGCTGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCG GCAGCGGCTCACGCAGCGCGTACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATC CTCGGGAGATGACGAAGACGGGGAGGACGAGGCTGAGGACACAGGTGTGGACACAGGGGC CCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGCTGGCCGTGCCGGCCGCCAA CACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTCCTGGCTGAA GAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCA GTGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGT GGAGAACAAGTTTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCC GCACCGGCCCATCCTGCAGGCGGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGA CGTGGAGTTCCACTGCAAGGTGTACAGTGACGCACAGCCCCACATCCAGTGGCTCAAGCA CGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACACCCTACGTTACCGTGCTCAA GACGGCGGGCGCTAACACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCACAACGTCAC CTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCA CTCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGG CAGTGTGTATGCAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGT GGCGGCTGTGACGCTCTGCCGCCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCAC CGTGCACAAGATCTCCCGCTTCCCGCTCAAGCGACAGGTGTCCCTGGAGTCCAACGCGTC CATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGTCCTCAGGGGAGGGCCCCAC GCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCTGTCTCGGGC CCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGA GGCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCT GAAAGACGATGCCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAA GATGATCGGGAAACACAAAAACATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCC CCTGTACGTGCTGGTGGAGTACGCGGCCAAGGGTAACCTGCGGGAGTTTCTGCGGGCGCG GCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGCCGCCCGAGGAGCAGCTCAC CTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTACTTGGCCTC CCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGT GATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAA GACGACCAACGGCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGT CTACACTCACCAGAGTGACGTCTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCT GGGGGGCTCCCCGTACCCCGGCATCCCTGTGGAGGAGCTCTTCAAGCTGCTGAAGGAGGG CCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGTACATGATCATGCGGGAGTG CTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGACCTGGACCG TGTCCTTACCGTGACGTCCACCGACGTAAAGGCGACACAGGAGGAGAACCGGGAGCTGAG GAGCAGGTGTGAGGAGCTCCACGGGAAGAACCTGGAACTGGGGAAGATCATGGACAGGTT CGAAGAGGTTGTGTACCAGGCCATGGAGGAAGTTCAGAAGCAGAAGGAACTTTCCAAAGC TGAAATCCAGAAAGTTCTAAAAGAAAAAGACCAACTTACCACAGATCTGAACTCCATGGA GAAGTCCTTCTCCGACCTCTTCAAGCGTTTTGAGAAACAGAAAGAGGTGATCGAGGGCTA CCGCAAGAACGAAGAGTCACTGAAGAAGTGCGTGGAGGATTACCTGGCAAGGATCACCCA GGAGGGCCAGAGGTACCAAGCCCTGAAGGCCCACGCGGAGGAGAAGCTGCAGCTGGCAAA CGAGGAGATCGCCCAGGTCCGGAGCAAGGCCCAGGCGGAAGCGTTGGCCCTCCAGGCCAG CCTGAGGAAGGAGCAGATGCGCATCCAGTCGCTGGAGAAGACAGTGGAGCAGAAGACTAA AGAGAACGAGGAGCTGACCAGGATCTGCGACGACCTCATCTCCAAGATGGAGAAGATCTG AFGFR3:TACC3 v3 >ATGGGCGCCCCTGCCTGCGCCCTCGCGCT (3376 base pairs)CTGCGTGGCCGTGGCCATCGTGGCCGGCGC (SEQ ID NO: 57)CTCCTCGGAGTCCTTGGGGACGGAGCAGCG CGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGAGCAGTTGGT CTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGG GCCCACTGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGG GCCCCAGCGGCTGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCG GCAGCGGCTCACGCAGCGCGTACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATC CTCGGGAGATGACGAAGACGGGGAGGACGAGGCTGAGGACACAGGTGTGGACACAGGGGC CCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGCTGGCCGTGCCGGCCGCCAA CACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTCCTGGCTGAA GAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCA GTGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGT GGAGAACAAGTTTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCC GCACCGGCCCATCCTGCAGGCGGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGA CGTGGAGTTCCACTGCAAGGTGTACAGTGACGCACAGCCCCACATCCAGTGGCTCAAGCA CGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACACCCTACGTTACCGTGCTCAA GACGGCGGGCGCTAACACCACCGACAAGGAGCTAGAGGTTCTCTCCTTGCACAACGTCAC CTTTGAGGACGCCGGGGAGTACACCTGCCTGGCGGGCAATTCTATTGGGTTTTCTCATCA CTCTGCGTGGCTGGTGGTGCTGCCAGCCGAGGAGGAGCTGGTGGAGGCTGACGAGGCGGG CAGTGTGTATGCAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCTGGTGGT GGCGGCTGTGACGCTCTGCCGCCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTCCCCCAC CGTGCACAAGATCTCCCGCTTCCCGCTCAAGCGACAGGTGTCCCTGGAGTCCAACGCGTC CATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGTCCTCAGGGGAGGGCCCCAC GCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCTGTCTCGGGC CCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCATGGCGGA GGCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAAGATGCT GAAAGACGATGCCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGATGATGAA GATGATCGGGAAACACAAAAACATCATCAACCTGCTGGGCGCCTGCACGCAGGGCGGGCC CCTGTACGTGCTGGTGGAGTACGCGGCCAAGGGTAACCTGCGGGAGTTTCTGCGGGCGCG GCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGCCGCCCGAGGAGCAGCTCAC CTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTACTTGGCCTC CCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGACAACGT GATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTACAAGAA GACGACCAACGGCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGACCGAGT CTACACTCACCAGAGTGACGTCTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTTCACGCT GGGGGGCTCCCCGTACCCCGGCATCCCTGTGGAGGAGCTCTTCAAGCTGCTGAAGGAGGG CCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGTACATGATCATGCGGGAGTG CTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGACCTGGACCG TGTCCTTACCGTGACGTCCACCGACGTGCCAGGCCCACCCCCAGGTGTTCCCGCGCCTGG GGGCCCACCCCTGTCCACCGGACCTATAGTGGACCTGCTCCAGTACAGCCAGAAGGACCT GGATGCAGTGGTAAAGGCGACACAGGAGGAGAACCGGGAGCTGAGGAGCAGGTGTGAGGA GCTCCACGGGAAGAACCTGGAACTGGGGAAGATCATGGACAGGTTCGAAGAGGTTGTGTA CCAGGCCATGGAGGAAGTTCAGAAGCAGAAGGAACTTTCCAAAGCTGAAATCCAGAAAGT TCTAAAAGAAAAAGACCAACTTACCACAGATCTGAACTCCATGGAGAAGTCCTTCTCCGA CCTCTTCAAGCGTTTTGAGAAACAGAAAGAGGTGATCGAGGGCTACCGCAAGAACGAAGA GTCACTGAAGAAGTGCGTGGAGGATTACCTGGCAAGGATCACCCAGGAGGGCCAGAGGTA CCAAGCCCTGAAGGCCCACGCGGAGGAGAAGCTGCAGCTGGCAAACGAGGAGATCGCCCA GGTCCGGAGCAAGGCCCAGGCGGAAGCGTTGGCCCTCCAGGCCAGCCTGAGGAAGGAGCA GATGCGCATCCAGTCGCTGGAGAAGACAGTGGAGCAGAAGACTAAAGAGAACGAGGAGCT GACCAGGATCTGCGACGACCTCATCTCCAAGATGGAGAAGATCTGA FGFR3 Intron: >ATGGGCGCCCCTGCCTGCGCCCTCGCGCT TACC3CTGCGTGGCCGTGGCCATCGTGGCCGGCGC (4463 base pairs)CTCCTCGGAGTCCTTGGGGACGGAGCAGCG (SEQ ID NO: 58)CGTCGTGGGGCGAGCGGCAGAAGTCCCGGG CCCAGAGCCCGGCCAGCAGGAGCAGTTGGTCTTCGGCAGCGGGGATGCTGTGGAGCTGAG CTGTCCCCCGCCCGGGGGTGGTCCCATGGGGCCCACTGTCTGGGTCAAGGATGGCACAGG GCTGGTGCCCTCGGAGCGTGTCCTGGTGGGGCCCCAGCGGCTGCAGGTGCTGAATGCCTC CCACGAGGACTCCGGGGCCTACAGCTGCCGGCAGCGGCTCACGCAGCGCGTACTGTGCCA CTTCAGTGTGCGGGTGACAGACGCTCCATCCTCGGGAGATGACGAAGACGGGGAGGACGA GGCTGAGGACACAGGTGTGGACACAGGGGCCCCTTACTGGACACGGCCCGAGCGGATGGA CAAGAAGCTGCTGGCCGTGCCGGCCGCCAACACCGTCCGCTTCCGCTGCCCAGCCGCTGG CAACCCCACTCCCTCCATCTCCTGGCTGAAGAACGGCAGGGAGTTCCGCGGCGAGCACCG CATTGGAGGCATCAAGCTGCGGCATCAGCAGTGGAGCCTGGTCATGGAAAGCGTGGTGCC CTCGGACCGCGGCAACTACACCTGCGTCGTGGAGAACAAGTTTGGCAGCATCCGGCAGAC GTACACGCTGGACGTGCTGGAGCGCTCCCCGCACCGGCCCATCCTGCAGGCGGGGCTGCC GGCCAACCAGACGGCGGTGCTGGGCAGCGACGTGGAGTTCCACTGCAAGGTGTACAGTGA CGCACAGCCCCACATCCAGTGGCTCAAGCACGTGGAGGTGAATGGCAGCAAGGTGGGCCC GGACGGCACACCCTACGTTACCGTGCTCAAGACGGCGGGCGCTAACACCACCGACAAGGA GCTAGAGGTTCTCTCCTTGCACAACGTCACCTTTGAGGACGCCGGGGAGTACACCTGCCT GGCGGGCAATTCTATTGGGTTTTCTCATCACTCTGCGTGGCTGGTGGTGCTGCCAGCCGA GGAGGAGCTGGTGGAGGCTGACGAGGCGGGCAGTGTGTATGCAGGCATCCTCAGCTACGG GGTGGGCTTCTTCCTGTTCATCCTGGTGGTGGCGGCTGTGACGCTCTGCCGCCTGCGCAG CCCCCCCAAGAAAGGCCTGGGCTCCCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAA GCGACAGGTGTCCCTGGAGTCCAACGCGTCCATGAGCTCCAACACACCACTGGTGCGCAT CGCAAGGCTGTCCTCAGGGGAGGGCCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCC TGCCGACCCCAAATGGGAGCTGTCTCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGA GGGCTGCTTCGGCCAGGTGGTCATGGCGGAGGCCATCGGCATTGACAAGGACCGGGCCGC CAAGCCTGTCACCGTAGCCGTGAAGATGCTGAAAGACGATGCCACTGACAAGGACCTGTC GGACCTGGTGTCTGAGATGGAGATGATGAAGATGATCGGGAAACACAAAAACATCATCAA CCTGCTGGGCGCCTGCACGCAGGGCGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAA GGGTAACCTGCGGGAGTTTCTGCGGGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGA CACCTGCAAGCCGCCCGAGGAGCAGCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCA GGTGGCCCGGGGCATGGAGTACTTGGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGC CCGCAATGTGCTGGTGACCGAGGACAACGTGATGAAGATCGCAGACTTCGGGCTGGCCCG GGACGTGCACAACCTCGACTACTACAAGAAGACGACCAACGGCCGGCTGCCCGTGAAGTG GATGGCGCCTGAGGCCTTGTTTGACCGAGTCTACACTCACCAGAGTGACGTCTGGTCCTT TGGGGTCCTGCTCTGGGAGATCTTCACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGT GGAGGAGCTCTTCAAGCTGCTGAAGGAGGGCCACCGCATGGACAAGCCCGCCAACTGCAC ACACGACCTGTACATGATCATGCGGGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCAC CTTCAAGCAGCTGGTGGAGGACCTGGACCGTGTCCTTACCGTGACGTCCACCGAC gtgag tgctggctctggcctggtgccacccgcctatgcccctccccctgccgtccccggccatcc tgccccccagagtgctgaggtgtggggcgggccttTCTGGCCCAGGTGCCCTGGCTGACC TGGACTGCTCAAGCTCTTCCCAGAGCCCAGGAAGTTCTGAGAACCAAATGGTGTCTCCAG GAAAAGTGTCTGGCAGCCCTGAGCAAGCCGTGGAGGAAAACCTTAGTTCCTATTCCTTAG ACAGAAGAGTGACACCCGCCTCTGAGACCCTAGAAGACCCTTGCAGGACAGAGTCCCAGC ACAAAGCGGAGACTCCGCACGGAGCCGAGGAAGAATGCAAAGCGGAGACTCCGCACGGAG CCGAGGAGGAATGCCGGCACGGTGGGGTCTGTGCTCCCGCAGCAGTGGCCACTTCGCCTC CTGGTGCAATCCCTAAGGAAGCCTGCGGAGGAGCACCCCTGCAGGGTCTGCCTGGCGAAG CCCTGGGCTGCCCTGCGGGTGTGGGCACCCCCGTGCCAGCAGATGGCACTCAGACCCTTA CCTGTGCACACACCTCTGCTCCTGAGAGCACAGCCCCAACCAACCACCTGGTGGCTGGCA GGGCCATGACCCTGAGTCCTCAGGAAGAAGTGGCTGCAGGCCAAATGGCCAGCTCCTCGA GGAGCGGACCTGTAAAACTAGAATTTGATGTATCTGATGGCGCCACCAGCAAAAGGGCAC CCCCACCAAGGAGACTGGGAGAGAGGTCCGGCCTCAAGCCTCCCTTGAGGAAAGCAGCAG TGAGGCAGCAAAAGGCCCCGCAGGAGGTGGAGGAGGACGACGGTAGGAGCGGAGCAGGAG AGGACCCCCCCATGCCAGCTTCTCGGGGCTCTTACCACCTCGACTGGGACAAAATGGATG ACCCAAACTTCATCCCGTTCGGAGGTGACACCAAGTCTGGTTGCAGTGAGGCCCAGCCCC CAGAAAGCCCTGAGACCAGGCTGGGCCAGCCAGCGGCTGAACAGTTGCATGCTGGGCCTG CCACGGAGGAGCCAGGTCCCTGTCTGAGCCAGCAGCTGCATTCAGCCTCAGCGGAGGACA CGCCTGTGGTGCAGTTGGCAGCCGAGACCCCAACAGCAGAGAGCAAGGAGAGAGCCTTGA ACTCTGCCAGCACCTCGCTTCCCACAAGCTGTCCAGGCAGTGAGCCAGTGCCCACCCATC AGCAGGGGCAGCCTGCCTTGGAGCTGAAAGAGGAGAGCTTCAGAGACCCCGCTGAGGTTC TAGGCACGGGCGCGGAGGTGGATTACCTGGAGCAGTTTGGAACTTCCTCGTTTAAGGAGT CGGCCTTGAGGAAGCAGTCCTTATACCTCAAGTTCGACCCCCTCCTGAGGGACAGTCCTG GTAGACCAGTGCCCGTGGCCACCGAGACCAGCAGCATGCACGGTGCAAATGAGACTCCCT CAGGACGTCCGCGGGAAGCCAAGCTTGTGGAGTTCGATTTCTTGGGAGCACTGGACATTC CTGTGCCAGGCCCACCCCCAGGTGTTCCCGCGCCTGGGGGCCCACCCCTGTCCACCGGAC CTATAGTGGACCTGCTCCAGTACAGCCAGAAGGACCTGGATGCAGTGGTAAAGGCGACAC AGGAGGAGAACCGGGAGCTGAGGAGCAGGTGTGAGGAGCTCCACGGGAAGAACCTGGAAC TGGGGAAGATCATGGACAGGTTCGAAGAGGTTGTGTACCAGGCCATGGAGGAAGTTCAGA AGCAGAAGGAACTTTCCAAAGCTGAAATCCAGAAAGTTCTAAAAGAAAAAGACCAACTTA CCACAGATCTGAACTCCATGGAGAAGTCCTTCTCCGACCTCTTCAAGCGTTTTGAGAAAC AGAAAGAGGTGATCGAGGGCTACCGCAAGAACGAAGAGTCACTGAAGAAGTGCGTGGAGG ATTACCTGGCAAGGATCACCCAGGAGGGCCAGAGGTACCAAGCCCTGAAGGCCCACGCGG AGGAGAAGCTGCAGCTGGCAAACGAGGAGATCGCCCAGGTCCGGAGCAAGGCCCAGGCGG AAGCGTTGGCCCTCCAGGCCAGCCTGAGGAAGGAGCAGATGCGCATCCAGTCGCTGGAGA AGACAGTGGAGCAGAAGACTAAAGAGAACGAGGAGCTGACCAGGATCTGCGACGACCTCA TCTCCAAGATGGAGAAGATCTGAFGFR3:BAIAP2L1 >ATGGGCGCCCCTGCCTGCGCCCTCGCGCT (3765 base pairs)CTGCGTGGCCGTGGCCATCGTGGCCGGCGC (SEQ ID NO: 59)CTCCTCGGAGTCCTTGGGGACGGAGCAGCG CGTCGTGGGGCGAGCGGCAGAAGTCCCGGGCCCAGAGCCCGGCCAGCAGGAGCAGTTGGT CTTCGGCAGCGGGGATGCTGTGGAGCTGAGCTGTCCCCCGCCCGGGGGTGGTCCCATGGG GCCCACTGTCTGGGTCAAGGATGGCACAGGGCTGGTGCCCTCGGAGCGTGTCCTGGTGGG GCCCCAGCGGCTGCAGGTGCTGAATGCCTCCCACGAGGACTCCGGGGCCTACAGCTGCCG GCAGCGGCTCACGCAGCGCGTACTGTGCCACTTCAGTGTGCGGGTGACAGACGCTCCATC CTCGGGAGATGACGAAGACGGGGAGGACGAGGCTGAGGACACAGGTGTGGACACAGGGGC CCCTTACTGGACACGGCCCGAGCGGATGGACAAGAAGCTGCTGGCCGTGCCGGCCGCCAA CACCGTCCGCTTCCGCTGCCCAGCCGCTGGCAACCCCACTCCCTCCATCTCCTGGCTGAA GAACGGCAGGGAGTTCCGCGGCGAGCACCGCATTGGAGGCATCAAGCTGCGGCATCAGCA GTGGAGCCTGGTCATGGAAAGCGTGGTGCCCTCGGACCGCGGCAACTACACCTGCGTCGT GGAGAACAAGTTTGGCAGCATCCGGCAGACGTACACGCTGGACGTGCTGGAGCGCTCCCC GCACCGGCCCATCCTGCAGGCGGGGCTGCCGGCCAACCAGACGGCGGTGCTGGGCAGCGA CGTGGAGTTCCACTGCAAGGTGTACAGTGACGCACAGCCCCACATCCAGTGGCTCAAGCA CGTGGAGGTGAATGGCAGCAAGGTGGGCCCGGACGGCACACCCTACGTTACCGTGCTCAA GTCCTGGATCAGTGAGAGTGTGGAGGCCGACGTGCGCCTCCGCCTGGCCAATGTGTCGGA GCGGGACGGGGGCGAGTACCTCTGTCGAGCCACCAATTTCATAGGCGTGGCCGAGAAGGC CTTTTGGCTGAGCGTTCACGGGCCCCGAGCAGCCGAGGAGGAGCTGGTGGAGGCTGACGA GGCGGGCAGTGTGTATGCAGGCATCCTCAGCTACGGGGTGGGCTTCTTCCTGTTCATCCT GGTGGTGGCGGCTGTGACGCTCTGCCGCCTGCGCAGCCCCCCCAAGAAAGGCCTGGGCTC CCCCACCGTGCACAAGATCTCCCGCTTCCCGCTCAAGCGACAGGTGTCCCTGGAGTCCAA CGCGTCCATGAGCTCCAACACACCACTGGTGCGCATCGCAAGGCTGTCCTCAGGGGAGGG CCCCACGCTGGCCAATGTCTCCGAGCTCGAGCTGCCTGCCGACCCCAAATGGGAGCTGTC TCGGGCCCGGCTGACCCTGGGCAAGCCCCTTGGGGAGGGCTGCTTCGGCCAGGTGGTCAT GGCGGAGGCCATCGGCATTGACAAGGACCGGGCCGCCAAGCCTGTCACCGTAGCCGTGAA GATGCTGAAAGACGATGCCACTGACAAGGACCTGTCGGACCTGGTGTCTGAGATGGAGAT GATGAAGATGATCGGGAAACACAAAAACATCATCAACCTGCTGGGCGCCTGCACGCAGGG CGGGCCCCTGTACGTGCTGGTGGAGTACGCGGCCAAGGGTAACCTGCGGGAGTTTCTGCG GGCGCGGCGGCCCCCGGGCCTGGACTACTCCTTCGACACCTGCAAGCCGCCCGAGGAGCA GCTCACCTTCAAGGACCTGGTGTCCTGTGCCTACCAGGTGGCCCGGGGCATGGAGTACTT GGCCTCCCAGAAGTGCATCCACAGGGACCTGGCTGCCCGCAATGTGCTGGTGACCGAGGA CAACGTGATGAAGATCGCAGACTTCGGGCTGGCCCGGGACGTGCACAACCTCGACTACTA CAAGAAGACGACCAACGGCCGGCTGCCCGTGAAGTGGATGGCGCCTGAGGCCTTGTTTGA CCGAGTCTACACTCACCAGAGTGACGTCTGGTCCTTTGGGGTCCTGCTCTGGGAGATCTT CACGCTGGGGGGCTCCCCGTACCCCGGCATCCCTGTGGAGGAGCTCTTCAAGCTGCTGAA GGAGGGCCACCGCATGGACAAGCCCGCCAACTGCACACACGACCTGTACATGATCATGCG GGAGTGCTGGCATGCCGCGCCCTCCCAGAGGCCCACCTTCAAGCAGCTGGTGGAGGACCT GGACCGTGTCCTTACCGTGACGTCCACCGACAATGTTATGGAACAGTTCAATCCTGGGCT GCGAAATTTAATAAACCTGGGGAAAAATTATGAGAAAGCTGTAAACGCTATGATCCTGGC AGGAAAAGCCTACTACGATGGAGTGGCCAAGATCGGTGAGATTGCCACTGGGTCCCCCGT GTCAACTGAACTGGGACATGTCCTCATAGAGATTTCAAGTACCCACAAGAAACTCAACGA GAGTCTTGATGAAAATTTTAAAAAATTCCACAAAGAGATTATCCATGAGCTGGAGAAGAA GATAGAACTTGACGTGAAATATATGAACGCAACTCTAAAAAGATACCAAACAGAACACAA GAATAAATTAGAGTCTTTGGAGAAATCCCAAGCTGAGTTGAAGAAGATCAGAAGGAAAAG CCAAGGAAGCCGAAACGCACTCAAATATGAACACAAAGAAATTGAGTATGTGGAGACCGT TACTTCTCGTCAGAGTGAAATCCAGAAATTCATTGCAGATGGTTGCAAAGAGGCTCTGCT TGAAGAGAAGAGGCGCTTCTGCTTTCTGGTTGATAAGCACTGTGGCTTTGCAAACCACAT ACATTATTATCACTTACAGTCTGCAGAACTACTGAATTCCAAGCTGCCTCGGTGGCAGGA GACCTGTGTTGATGCCATCAAAGTGCCAGAGAAAATCATGAATATGATCGAAGAAATAAA GACCCCAGCCTCTACCCCCGTGTCTGGAACTCCTCAGGCTTCACCCATGATCGAGAGAAG CAATGTGGTTAGGAAAGATTACGACACCCTTTCTAAATGCTCACCAAAGATGCCCCCCGC TCCTTCAGGCAGAGCATATACCAGTCCCTTGATCGATATGTTTAATAACCCAGCCACGGC TGCCCCGAATTCACAAAGGGTAAATAATTCAACAGGTACTTCCGAAGATCCCAGTTTACA GCGATCAGTTTCGGTTGCAACGGGACTGAACATGATGAAGAAGCAGAAAGTGAAGACCAT CTTCCCGCACACTGCGGGCTCCAACAAGACCTTACTCAGCTTTGCACAGGGAGATGTCAT CACGCTGCTCATCCCCGAGGAGAAGGATGGCTGGCTCTATGGAGAACACGACGTGTCCAA GGCGAGGGGTTGGTTCCCGTCGTCGTACACGAAGTTGCTGGAAGAAAATGAGACAGAAGC AGTGACCGTGCCCACGCCAAGCCCCACACCAGTGAGAAGCATCAGCACCGTGAACTTGTC TGAGAATAGCAGTGTTGTCATCCCCCCACCCGACTACTTGGAATGCTTGTCCATGGGGGC AGCTGCCGACAGGAGAGCAGATTCGGCCAGGACGACATCCACCTTTAAGGCCCCAGCGTC CAAGCCCGAGACCGCGGCTCCTAACGATGCCAACGGGACTGCAAAGCCGCCTTTTCTCAG CGGAGAAAACCCCTTTGCCACTGTGAAACTCCGCCCGACTGTGACGAATGATCGCTCGGC ACCCATCATTCGATGAFGFR2:BICC1 >ATGGTCAGCTGGGGTCGTTTCATCTGCCT (5830 base pairs)GGTCGTGGTCACCATGGCAACCTTGTCCCT (SEQ ID NO: 60)GGCCCGGCCCTCCTTCAGTTTAGTTGAGGA TACCACATTAGAGCCAGAAGAGCCACCAACCAAATACCAAATCTCTCAACCAGAAGTGTA CGTGGCTGCGCCAGGGGAGTCGCTAGAGGTGCGCTGCCTGTTGAAAGATGCCGCCGTGAT CAGTTGGACTAAGGATGGGGTGCACTTGGGGCCCAACAATAGGACAGTGCTTATTGGGGA GTACTTGCAGATAAAGGGCGCCACGCCTAGAGACTCCGGCCTCTATGCTTGTACTGCCAG TAGGACTGTAGACAGTGAAACTTGGTACTTCATGGTGAATGTCACAGATGCCATCTCATC CGGAGATGATGAGGATGACACCGATGGTGCGGAAGATTTTGTCAGTGAGAACAGTAACAA CAAGAGAGCACCATACTGGACCAACACAGAAAAGATGGAAAAGCGGCTCCATGCTGTGCC TGCGGCCAACACTGTCAAGTTTCGCTGCCCAGCCGGGGGGAACCCAATGCCAACCATGCG GTGGCTGAAAAACGGGAAGGAGTTTAAGCAGGAGCATCGCATTGGAGGCTACAAGGTACG AAACCAGCACTGGAGCCTCATTATGGAAAGTGTGGTCCCATCTGACAAGGGAAATTATAC CTGTGTAGTGGAGAATGAATACGGGTCCATCAATCACACGTACCACCTGGATGTTGTGGA GCGATCGCCTCACCGGCCCATCCTCCAAGCCGGACTGCCGGCAAATGCCTCCACAGTGGT CGGAGGAGACGTAGAGTTTGTCTGCAAGGTTTACAGTGATGCCCAGCCCCACATCCAGTG GATCAAGCACGTGGAAAAGAACGGCAGTAAATACGGGCCCGACGGGCTGCCCTACCTCAA GGTTCTCAAGGCCGCCGGTGTTAACACCACGGACAAAGAGATTGAGGTTCTCTATATTCG GAATGTAACTTTTGAGGACGCTGGGGAATATACGTGCTTGGCGGGTAATTCTATTGGGAT ATCCTTTCACTCTGCATGGTTGACAGTTCTGCCAGCGCCTGGAAGAGAAAAGGAGATTAC AGCTTCCCCAGACTACCTGGAGATAGCCATTTACTGCATAGGGGTCTTCTTAATCGCCTG TATGGTGGTAACAGTCATCCTGTGCCGAATGAAGAACACGACCAAGAAGCCAGACTTCAG CAGCCAGCCGGCTGTGCACAAGCTGACCAAACGTATCCCCCTGCGGAGACAGGTAACAGT TTCGGCTGAGTCCAGCTCCTCCATGAACTCCAACACCCCGCTGGTGAGGATAACAACACG CCTCTCTTCAACGGCAGACACCCCCATGCTGGCAGGGGTCTCCGAGTATGAACTTCCAGA GGACCCAAAATGGGAGTTTCCAAGAGATAAGCTGACACTGGGCAAGCCCCTGGGAGAAGG TTGCTTTGGGCAAGTGGTCATGGCGGAAGCAGTGGGAATTGACAAAGACAAGCCCAAGGA GGCGGTCACCGTGGCCGTGAAGATGTTGAAAGATGATGCCACAGAGAAAGACCTTTCTGA TCTGGTGTCAGAGATGGAGATGATGAAGATGATTGGGAAACACAAGAATATCATAAATCT TCTTGGAGCCTGCACACAGGATGGGCCTCTCTATGTCATAGTTGAGTATGCCTCTAAAGG CAACCTCCGAGAATACCTCCGAGCCCGGAGGCCACCCGGGATGGAGTACTCCTATGACAT TAACCGTGTTCCTGAGGAGCAGATGACCTTCAAGGACTTGGTGTCATGCACCTACCAGCT GGCCAGAGGCATGGAGTACTTGGCTTCCCAAAAATGTATTCATCGAGATTTAGCAGCCAG AAATGTTTTGGTAACAGAAAACAATGTGATGAAAATAGCAGACTTTGGACTCGCCAGAGA TATCAACAATATAGACTATTACAAAAAGACCACCAATGGGCGGCTTCCAGTCAAGTGGAT GGCTCCAGAAGCCCTGTTTGATAGAGTATACACTCATCAGAGTGATGTCTGGTCCTTCGG GGTGTTAATGTGGGAGATCTTCACTTTAGGGGGCTCGCCCTACCCAGGGATTCCCGTGGA GGAACTTTTTAAGCTGCTGAAGGAAGGACACAGAATGGATAAGCCAGCCAACTGCACCAA CGAACTGTACATGATGATGAGGGACTGTTGGCATGCAGTGCCCTCCCAGAGACCAACGTT CAAGCAGTTGGTAGAAGACTTGGATCGAATTCTCACTCTCACAACCAATGAGATCATGGA GGAAACAAATACGCAGATTGCTTGGCCATCAAAACTGAAGATCGGAGCCAAATCCAAGAA AGATCCCCATATTAAGGTTTCTGGAAAGAAAGAAGATGTTAAAGAAGCCAAGGAAATGAT CATGTCTGTCTTAGACACAAAAAGCAATCGAGTCACACTGAAGATGGATGTTTCACATAC AGAACATTCACATGTAATCGGCAAAGGTGGCAACAATATTAAAAAAGTGATGGAAGAAAC CGGATGCCATATCCACTTTCCAGATTCCAACAGGAATAACCAAGCAGAAAAAAGCAACCA GGTATCTATAGCGGGACAACCAGCAGGAGTAGAATCTGCCCGAGTTAGAATTCGGGAGCT GCTTCCTTTGGTGCTGATGTTTGAGCTACCAATTGCTGGAATTCTTCAACCGGTTCCTGA TCCTAATTCCCCCTCTATTCAGCATATATCACAAACGTACAATATTTCAGTATCATTTAA ACAGCGTTCCCGAATGTATGGTGCTACTGTCATAGTACGAGGGTCTCAGAATAACACTAG TGCTGTGAAGGAAGGAACTGCCATGCTGTTAGAACATCTTGCTGGGAGCTTAGCATCAGC TATTCCTGTGAGCACACAACTAGATATTGCAGCTCAACATCATCTCTTTATGATGGGTCG AAATGGGAGCAACATCAAACATATCATGCAGAGAACAGGTGCTCAGATCCACTTTCCTGA TCCCAGTAATCCACAAAAGAAATCTACCGTCTACCTCCAGGGCACCATTGAGTCTGTCTG TCTTGCAAGGCAATATCTCATGGGTTGTCTTCCTCTTGTGTTGATGTTTGATATGAAGGA AGAAATTGAAGTAGATCCACAATTCATTGCGCAGTTGATGGAACAGCTTGATGTCTTCAT CAGTATTAAACCAAAGCCCAAACAGCCAAGCAAGTCTGTGATTGTGAAAAGTGTTGAGCG AAATGCCTTAAATATGTATGAAGCAAGGAAATGTCTCCTCGGACTTGAAAGCAGTGGGGT TACCATAGCAACCAGTCCATCCCCAGCATCCTGCCCTGCCGGCCTGGCATGTCCCAGCCT GGATATCTTAGCTTCAGCAGGCCTTGGACTCACTGGACTAGGTCTTTTGGGACCCACCAC CTTATCTCTGAACACTTCAACAACCCCAAACTCACTCTTGAATGCTCTTAATAGCTCAGT CAGTCCTTTGCAAAGTCCAAGTTCTGGTACACCCAGCCCCACATTATGGGCACCCCCACT TGCTAATACTTCAAGTGCCACAGGTTTTTCTGCTATACCACACCTTATGATTCCATCTAC TGCCCAAGCCACATTAACTAATATTTTGTTGTCTGGAGTGCCCACCTATGGGCACACAGC TCCATCTCCCCCTCCTGGCTTGACTCCTGTTGATGTCCATATCAACAGTATGCAGACCGA AGGCAAAAAAATCTCTGCTGCTTTAAATGGACATGCACAGTCTCCAGATATAAAATATGG TGCAATATCCACTTCATCACTTGGAGAAAAAGTGCTGAGTGCAAATCACGGGGATCCGTC CATCCAGACAAGTGGGTCTGAGCAGACATCTCCCAAATCAAGCCCCACTGAAGGTTGTAA TGATGCTTTTGTTGAAGTAGGCATGCCTCGAAGTCCTTCCCATTCTGGGAATGCTGGTGA CTTGAAACAGATGATGTGTCCCTCCAAGGTTTCCTGTGCCAAAAGGCAGACAGTGGAACT ATTGCAAGGCACGAAAAACTCACACTTACACAGCACTGACAGGTTGCTCTCAGACCCTGA ACTGAGTGCTACCGAAAGCCCTTTGGCTGACAAGAAGGCTCCAGGGAGTGAGCGCGCTGC AGAGAGGGCAGCAGCTGCCCAGCAAAACTCCGAAAGGGCCCACCTTGCTCCACGGTCATC ATATGTCAACATGCAGGCATTTGACTATGAACAGAAGAAGCTATTAGCCACCAAAGCTAT GTTAAAGAAACCAGTGGTGACGGAGGTCAGAACGCCCACAAATACCTGGAGTGGCCTGGG TTTTTCTAAATCCATGCCAGCTGAAACTATCAAGGAGTTGAGAAGGGCCAATCATGTGTC CTATAAGCCCACAATGACAACCACTTATGAGGGCTCATCCATGTCCCTTTCACGGTCCAA CAGTCGTGAGCACTTGGGAGGTGGAAGCGAATCTGATAACTGGAGAGACCGAAATGGAAT TGGACCTGGAAGTCATAGTGAATTTGCAGCTTCTATTGGCAGCCCTAAGCGTAAACAAAA CAAATCAACGGAACACTATCTCAGCAGTAGCAATTACATGGACTGCATTTCCTCGCTGAC AGGAAGCAATGGCTGTAACTTAAATAGCTCTTTCAAAGGTTCTGACCTCCCTGAGCTCTT CAGCAAACTGGGCCTGGGCAAATACACAGATGTTTTCCAGCAACAAGAGATCGATCTTCA GACATTCCTCACTCTCACAGATCAGGATCTGAAGGAGCTGGGAATAACTACTTTTGGTGC CAGGAGGAAAATGCTGCTTGCAATTTCAGAACTAAATAAAAACCGAAGAAAGCTTTTTGA ATCGCCAAATGCACGCACCTCTTTCCTGGAAGGTGGAGCGAGTGGAAGGCTACCCCGTCA GTATCACTCAGACATTGCTAGTGTCAGTGG CCGCTGGTAGFGFR2:AFF3 >ATGGTCAGCTGGGGTCGTTTCATCTGCCT (5109 base pairs)GGTCGTGGTCACCATGGCAACCTTGTCCCT (SEQ ID NO: 61)GGCCCGGCCCTCCTTCAGTTTAGTTGAGGA TACCACATTAGAGCCAGAAGAGCCACCAACCAAATACCAAATCTCTCAACCAGAAGTGTA CGTGGCTGCGCCAGGGGAGTCGCTAGAGGTGCGCTGCCTGTTGAAAGATGCCGCCGTGAT CAGTTGGACTAAGGATGGGGTGCACTTGGGGCCCAACAATAGGACAGTGCTTATTGGGGA GTACTTGCAGATAAAGGGCGCCACGCCTAGAGACTCCGGCCTCTATGCTTGTACTGCCAG TAGGACTGTAGACAGTGAAACTTGGTACTTCATGGTGAATGTCACAGATGCCATCTCATC CGGAGATGATGAGGATGACACCGATGGTGCGGAAGATTTTGTCAGTGAGAACAGTAACAA CAAGAGAGCACCATACTGGACCAACACAGAAAAGATGGAAAAGCGGCTCCATGCTGTGCC TGCGGCCAACACTGTCAAGTTTCGCTGCCCAGCCGGGGGGAACCCAATGCCAACCATGCG GTGGCTGAAAAACGGGAAGGAGTTTAAGCAGGAGCATCGCATTGGAGGCTACAAGGTACG AAACCAGCACTGGAGCCTCATTATGGAAAGTGTGGTCCCATCTGACAAGGGAAATTATAC CTGTGTAGTGGAGAATGAATACGGGTCCATCAATCACACGTACCACCTGGATGTTGTGGA GCGATCGCCTCACCGGCCCATCCTCCAAGCCGGACTGCCGGCAAATGCCTCCACAGTGGT CGGAGGAGACGTAGAGTTTGTCTGCAAGGTTTACAGTGATGCCCAGCCCCACATCCAGTG GATCAAGCACGTGGAAAAGAACGGCAGTAAATACGGGCCCGACGGGCTGCCCTACCTCAA GGTTCTCAAGGCCGCCGGTGTTAACACCACGGACAAAGAGATTGAGGTTCTCTATATTCG GAATGTAACTTTTGAGGACGCTGGGGAATATACGTGCTTGGCGGGTAATTCTATTGGGAT ATCCTTTCACTCTGCATGGTTGACAGTTCTGCCAGCGCCTGGAAGAGAAAAGGAGATTAC AGCTTCCCCAGACTACCTGGAGATAGCCATTTACTGCATAGGGGTCTTCTTAATCGCCTG TATGGTGGTAACAGTCATCCTGTGCCGAATGAAGAACACGACCAAGAAGCCAGACTTCAG CAGCCAGCCGGCTGTGCACAAGCTGACCAAACGTATCCCCCTGCGGAGACAGGTAACAGT TTCGGCTGAGTCCAGCTCCTCCATGAACTCCAACACCCCGCTGGTGAGGATAACAACACG CCTCTCTTCAACGGCAGACACCCCCATGCTGGCAGGGGTCTCCGAGTATGAACTTCCAGA GGACCCAAAATGGGAGTTTCCAAGAGATAAGCTGACACTGGGCAAGCCCCTGGGAGAAGG TTGCTTTGGGCAAGTGGTCATGGCGGAAGCAGTGGGAATTGACAAAGACAAGCCCAAGGA GGCGGTCACCGTGGCCGTGAAGATGTTGAAAGATGATGCCACAGAGAAAGACCTTTCTGA TCTGGTGTCAGAGATGGAGATGATGAAGATGATTGGGAAACACAAGAATATCATAAATCT TCTTGGAGCCTGCACACAGGATGGGCCTCTCTATGTCATAGTTGAGTATGCCTCTAAAGG CAACCTCCGAGAATACCTCCGAGCCCGGAGGCCACCCGGGATGGAGTACTCCTATGACAT TAACCGTGTTCCTGAGGAGCAGATGACCTTCAAGGACTTGGTGTCATGCACCTACCAGCT GGCCAGAGGCATGGAGTACTTGGCTTCCCAAAAATGTATTCATCGAGATTTAGCAGCCAG AAATGTTTTGGTAACAGAAAACAATGTGATGAAAATAGCAGACTTTGGACTCGCCAGAGA TATCAACAATATAGACTATTACAAAAAGACCACCAATGGGCGGCTTCCAGTCAAGTGGAT GGCTCCAGAAGCCCTGTTTGATAGAGTATACACTCATCAGAGTGATGTCTGGTCCTTCGG GGTGTTAATGTGGGAGATCTTCACTTTAGGGGGCTCGCCCTACCCAGGGATTCCCGTGGA GGAACTTTTTAAGCTGCTGAAGGAAGGACACAGAATGGATAAGCCAGCCAACTGCACCAA CGAACTGTACATGATGATGAGGGACTGTTGGCATGCAGTGCCCTCCCAGAGACCAACGTT CAAGCAGTTGGTAGAAGACTTGGATCGAATTCTCACTCTCACAACCAATGAGGAGAGTAG ATCTGGAGAAACCAACAGCTGTGTTGAAGAAATAATCCGGGAGATGACCTGGCTTCCACC ACTTTCTGCTATTCAAGCACCTGGCAAAGTGGAACCAACCAAATTTCCATTTCCAAATAA GGACTCTCAGCTTGTATCCTCTGGACACAATAATCCAAAGAAAGGTGATGCAGAGCCAGA GAGTCCAGACAGTGGCACATCGAATACATCAATGCTGGAAGATGACCTTAAGCTAAGCAG TGATGAAGAGGAGAATGAACAGCAGGCAGCTCAGAGAACGGCTCTCCGCGCTCTCTCTGA CAGCGCCGTGGTCCAGCAGCCCAACTGCAGAACCTCGGTGCCTTCCAGCAAGGGCAGCAG CAGCAGCAGCAGCAGCGGCAGCAGCAGCTCCTCCAGCGACTCAGAGAGCAGCTCCGGATC TGACTCGGAGACCGAGAGCAGCTCCAGCGAGAGTGAGGGCAGCAAGCCCCCCCACTTCTC CAGCCCCGAGGCTGAACCGGCATCCTCTAACAAGTGGCAGCTGGATAAATGGCTAAACAA AGTTAATCCCCACAAGCCTCCTATTCTGATCCAAAATGAAAGCCACGGGTCAGAGAGCAA TCAGTACTACAACCCGGTGAAAGAGGACGTCCAGGACTGTGGGAAAGTCCCCGACGTTTG CCAGCCCAGCCTGAGAGAGAAGGAGATCAAGAGCACTTGCAAGGAGGAGCAAAGGCCAAG GACAGCCAACAAGGCCCCTGGGAGTAAAGGCGTGAAGCAGAAGTCCCCGCCCGCGGCCGT GGCCGTGGCGGTGAGCGCAGCCGCCCCGCCACCCGCAGTGCCCTGTGCGCCCGCGGAGAA CGCGCCCGCGCCTGCCCGGAGGTCCGCGGGCAAGAAGCCCACCAGGCGCACCGAGAGGAC CTCAGCCGGGGACGGCGCCAACTGCCACCGGCCCGAGGAGCCCGCGGCCGCGGACGCGCT GGGGACGAGCGTGGTGGTCCCCCCGGAGCCCACCAAAACCAGGCCCTGTGGCAACAACAG AGCGAGCCACCGCAAGGAGCTGCGCTCCTCCGTGACCTGCGAGAAGCGCCGCACGCGGGG GCTAAGCAGGATCGTCCCCAAATCCAAGGAGTTCATTGAGACAGAGTCGTCATCTTCATC CTCCTCCTCGGACTCCGACCTGGAGTCCGAGCAGGAGGAGTACCCTCTGTCCAAAGCACA GACCGTGGCTGCCTCTGCCTCCTCCGGGAATGATCAGAGGCTGAAGGAGGCCGCTGCCAA CGGGGGCAGTGGTCCTAGGGCCCCTGTAGGCTCCATCAACGCCAGGACCACCAGTGACAT CGCCAAGGAGCTGGAGGAGCAGTTCTACACACTGGTCCCCTTTGGCCGGAACGAACTTCT CTCCCCTCTAAAGGACAGTGATGAGATCAGGTCTCTCTGGGTCAAAATCGACCTGACCCT CCTGTCCAGGATCCCAGAACACCTGCCCCAGGAGCCAGGGGTATTGAGCGCCCCTGCCAC CAAGGACTCTGAGAGCGCACCGCCCAGCCACACCTCGGACACACCTGCAGAAAAGGCTTT GCCAAAATCCAAGAGGAAACGCAAGTGTGACAACGAAGACGACTACAGGGAGATCAAGAA GTCCCAGGGAGAGAAAGACAGCTCTTCAAGACTGGCCACCTCCACCAGTAATACTTTGTC TGCAAACCACTGCAACATGAACATCAACAGTGTGGCAATACCAATAAATAAAAATGAAAA AATGCTTCGGTCGCCCATCTCACCCCTCTCTGATGCATCTAAACACAAATACACCAGCGA GGACTTAACTTCTTCCAGCCGACCTAATGGCAACAGTTTGTTTACTTCAGCCTCTTCCAG CAAAAAGCCTAAGGCCGACAGCCAGCTGCAGCCTCACGGCGGAGACCTCACGAAAGCAGC TCACAACAATTCTGAAAACATTCCCCTCCACAAGTCACGGCCGCAGACGAAGCCGTGGTC TCCAGGCTCCAACGGCCACAGGGACTGCAAGAGGCAGAAACTTGTCTTCGATGATATGCC TCGCAGTGCCGATTATTTTATGCAAGAAGCTAAACGAATGAAGCATAAAGCAGATGCAAT GGTGGAAAAGTTTGGAAAGGCTTTGAACTATGCTGAAGCAGCATTGTCGTTTATCGAGTG TGGAAATGCAATGGAACAAGGCCCCATGGAATCCAAATCTCCTTATACGATGTATTCAGA AACAGTAGAGCTCATCAGGTATGCTATGAGACTAAAAACCCACTCAGGCCCCAATGCCAC ACCAGAAGACAAACAACTGGCTGCATTATGTTACCGATGCCTGGCCCTCCTGTACTGGCG GATGTTTCGACTCAAAAGGGACCACGCTGTAAAGTATTCAAAAGCACTAATCGACTATTT CAAGAACTCATCTAAAGCCGCCCAAGCCCCATCTCCGTGGGGGGCCAGTGGAAAGAGCAC TGGAACCCCATCCCCCATGTCTCCCAACCCCTCTCCCGCCAGCTCCGTGGGGTCTCAGGG CAGCCTCTCCAACGCCAGCGCCCTGTCCCCGTCGACCATCGTCAGCATCCCACAGCGCAT CCACCAGATGGCGGCCAACCACGTCAGCATCACCAACAGCATCCTGCACAGCTACGACTA CTGGGAGATGGCCGACAACCTGGCCAAGGAAAACCGAGAATTCTTCAACGACCTGGATCT GCTCATGGGGCCGGTCACCCTGCACAGCAGCATGGAGCACCTGGTCCAGTACTCCCAACA GGGCCTGCACTGGCTGCGGAACAGCGCCCA CCTGTCATAGFGFR2:CASP7 >ATGGTCAGCTGGGGTCGTTTCATCTGCCT (3213 base pairs)GGTCGTGGTCACCATGGCAACCTTGTCCCT (SEQ ID NO: 62)GGCCCGGCCCTCCTTCAGTTTAGTTGAGGA TACCACATTAGAGCCAGAAGAGCCACCAACCAAATACCAAATCTCTCAACCAGAAGTGTA CGTGGCTGCGCCAGGGGAGTCGCTAGAGGTGCGCTGCCTGTTGAAAGATGCCGCCGTGAT CAGTTGGACTAAGGATGGGGTGCACTTGGGGCCCAACAATAGGACAGTGCTTATTGGGGA GTACTTGCAGATAAAGGGCGCCACGCCTAGAGACTCCGGCCTCTATGCTTGTACTGCCAG TAGGACTGTAGACAGTGAAACTTGGTACTTCATGGTGAATGTCACAGATGCCATCTCATC CGGAGATGATGAGGATGACACCGATGGTGCGGAAGATTTTGTCAGTGAGAACAGTAACAA CAAGAGAGCACCATACTGGACCAACACAGAAAAGATGGAAAAGCGGCTCCATGCTGTGCC TGCGGCCAACACTGTCAAGTTTCGCTGCCCAGCCGGGGGGAACCCAATGCCAACCATGCG GTGGCTGAAAAACGGGAAGGAGTTTAAGCAGGAGCATCGCATTGGAGGCTACAAGGTACG AAACCAGCACTGGAGCCTCATTATGGAAAGTGTGGTCCCATCTGACAAGGGAAATTATAC CTGTGTAGTGGAGAATGAATACGGGTCCATCAATCACACGTACCACCTGGATGTTGTGGA GCGATCGCCTCACCGGCCCATCCTCCAAGCCGGACTGCCGGCAAATGCCTCCACAGTGGT CGGAGGAGACGTAGAGTTTGTCTGCAAGGTTTACAGTGATGCCCAGCCCCACATCCAGTG GATCAAGCACGTGGAAAAGAACGGCAGTAAATACGGGCCCGACGGGCTGCCCTACCTCAA GGTTCTCAAGGCCGCCGGTGTTAACACCACGGACAAAGAGATTGAGGTTCTCTATATTCG GAATGTAACTTTTGAGGACGCTGGGGAATATACGTGCTTGGCGGGTAATTCTATTGGGAT ATCCTTTCACTCTGCATGGTTGACAGTTCTGCCAGCGCCTGGAAGAGAAAAGGAGATTAC AGCTTCCCCAGACTACCTGGAGATAGCCATTTACTGCATAGGGGTCTTCTTAATCGCCTG TATGGTGGTAACAGTCATCCTGTGCCGAATGAAGAACACGACCAAGAAGCCAGACTTCAG CAGCCAGCCGGCTGTGCACAAGCTGACCAAACGTATCCCCCTGCGGAGACAGGTAACAGT TTCGGCTGAGTCCAGCTCCTCCATGAACTCCAACACCCCGCTGGTGAGGATAACAACACG CCTCTCTTCAACGGCAGACACCCCCATGCTGGCAGGGGTCTCCGAGTATGAACTTCCAGA GGACCCAAAATGGGAGTTTCCAAGAGATAAGCTGACACTGGGCAAGCCCCTGGGAGAAGG TTGCTTTGGGCAAGTGGTCATGGCGGAAGCAGTGGGAATTGACAAAGACAAGCCCAAGGA GGCGGTCACCGTGGCCGTGAAGATGTTGAAAGATGATGCCACAGAGAAAGACCTTTCTGA TCTGGTGTCAGAGATGGAGATGATGAAGATGATTGGGAAACACAAGAATATCATAAATCT TCTTGGAGCCTGCACACAGGATGGGCCTCTCTATGTCATAGTTGAGTATGCCTCTAAAGG CAACCTCCGAGAATACCTCCGAGCCCGGAGGCCACCCGGGATGGAGTACTCCTATGACAT TAACCGTGTTCCTGAGGAGCAGATGACCTTCAAGGACTTGGTGTCATGCACCTACCAGCT GGCCAGAGGCATGGAGTACTTGGCTTCCCAAAAATGTATTCATCGAGATTTAGCAGCCAG AAATGTTTTGGTAACAGAAAACAATGTGATGAAAATAGCAGACTTTGGACTCGCCAGAGA TATCAACAATATAGACTATTACAAAAAGACCACCAATGGGCGGCTTCCAGTCAAGTGGAT GGCTCCAGAAGCCCTGTTTGATAGAGTATACACTCATCAGAGTGATGTCTGGTCCTTCGG GGTGTTAATGTGGGAGATCTTCACTTTAGGGGGCTCGCCCTACCCAGGGATTCCCGTGGA GGAACTTTTTAAGCTGCTGAAGGAAGGACACAGAATGGATAAGCCAGCCAACTGCACCAA CGAACTGTACATGATGATGAGGGACTGTTGGCATGCAGTGCCCTCCCAGAGACCAACGTT CAAGCAGTTGGTAGAAGACTTGGATCGAATTCTCACTCTCACAACCAATGAGATGGCAGA TGATCAGGGCTGTATTGAAGAGCAGGGGGTTGAGGATTCAGCAAATGAAGATTCAGTGGA TGCTAAGCCAGACCGGTCCTCGTTTGTACCGTCCCTCTTCAGTAAGAAGAAGAAAAATGT CACCATGCGATCCATCAAGACCACCCGGGACCGAGTGCCTACATATCAGTACAACATGAA TTTTGAAAAGCTGGGCAAATGCATCATAATAAACAACAAGAACTTTGATAAAGTGACAGG TATGGGCGTTCGAAACGGAACAGACAAAGATGCCGAGGCGCTCTTCAAGTGCTTCCGAAG CCTGGGTTTTGACGTGATTGTCTATAATGACTGCTCTTGTGCCAAGATGCAAGATCTGCT TAAAAAAGCTTCTGAAGAGGACCATACAAATGCCGCCTGCTTCGCCTGCATCCTCTTAAG CCATGGAGAAGAAAATGTAATTTATGGGAAAGATGGTGTCACACCAATAAAGGATTTGAC AGCCCACTTTAGGGGGGATAGATGCAAAACCCTTTTAGAGAAACCCAAACTCTTCTTCAT TCAGGCTTGCCGAGGGACCGAGCTTGATGATGGCATCCAGGCCGACTCGGGGCCCATCAA TGACACAGATGCTAATCCTCGATACAAGATCCCAGTGGAAGCTGACTTCCTCTTCGCCTA TTCCACGGTTCCAGGCTATTACTCGTGGAGGAGCCCAGGAAGAGGCTCCTGGTTTGTGCA AGCCCTCTGCTCCATCCTGGAGGAGCACGGAAAAGACCTGGAAATCATGCAGATCCTCAC CAGGGTGAATGACAGAGTTGCCAGGCACTTTGAGTCTCAGTCTGATGACCCACACTTCCA TGAGAAGAAGCAGATCCCCTGTGTGGTCTCCATGCTCACCAAGGAACTCTACTTCAGTCA ATAGFGFR2:CCDC6 >ATGGTCAGCTGGGGTCGTTTCATCTGCCT (3423 base pairs)GGTCGTGGTCACCATGGCAACCTTGTCCCT (SEQ ID NO: 63)GGCCCGGCCCTCCTTCAGTTTAGTTGAGGA TACCACATTAGAGCCAGAAGAGCCACCAACCAAATACCAAATCTCTCAACCAGAAGTGTA CGTGGCTGCGCCAGGGGAGTCGCTAGAGGTGCGCTGCCTGTTGAAAGATGCCGCCGTGAT CAGTTGGACTAAGGATGGGGTGCACTTGGGGCCCAACAATAGGACAGTGCTTATTGGGGA GTACTTGCAGATAAAGGGCGCCACGCCTAGAGACTCCGGCCTCTATGCTTGTACTGCCAG TAGGACTGTAGACAGTGAAACTTGGTACTTCATGGTGAATGTCACAGATGCCATCTCATC CGGAGATGATGAGGATGACACCGATGGTGCGGAAGATTTTGTCAGTGAGAACAGTAACAA CAAGAGAGCACCATACTGGACCAACACAGAAAAGATGGAAAAGCGGCTCCATGCTGTGCC TGCGGCCAACACTGTCAAGTTTCGCTGCCCAGCCGGGGGGAACCCAATGCCAACCATGCG GTGGCTGAAAAACGGGAAGGAGTTTAAGCAGGAGCATCGCATTGGAGGCTACAAGGTACG AAACCAGCACTGGAGCCTCATTATGGAAAGTGTGGTCCCATCTGACAAGGGAAATTATAC CTGTGTAGTGGAGAATGAATACGGGTCCATCAATCACACGTACCACCTGGATGTTGTGGA GCGATCGCCTCACCGGCCCATCCTCCAAGCCGGACTGCCGGCAAATGCCTCCACAGTGGT CGGAGGAGACGTAGAGTTTGTCTGCAAGGTTTACAGTGATGCCCAGCCCCACATCCAGTG GATCAAGCACGTGGAAAAGAACGGCAGTAAATACGGGCCCGACGGGCTGCCCTACCTCAA GGTTCTCAAGGCCGCCGGTGTTAACACCACGGACAAAGAGATTGAGGTTCTCTATATTCG GAATGTAACTTTTGAGGACGCTGGGGAATATACGTGCTTGGCGGGTAATTCTATTGGGAT ATCCTTTCACTCTGCATGGTTGACAGTTCTGCCAGCGCCTGGAAGAGAAAAGGAGATTAC AGCTTCCCCAGACTACCTGGAGATAGCCATTTACTGCATAGGGGTCTTCTTAATCGCCTG TATGGTGGTAACAGTCATCCTGTGCCGAATGAAGAACACGACCAAGAAGCCAGACTTCAG CAGCCAGCCGGCTGTGCACAAGCTGACCAAACGTATCCCCCTGCGGAGACAGGTAACAGT TTCGGCTGAGTCCAGCTCCTCCATGAACTCCAACACCCCGCTGGTGAGGATAACAACACG CCTCTCTTCAACGGCAGACACCCCCATGCTGGCAGGGGTCTCCGAGTATGAACTTCCAGA GGACCCAAAATGGGAGTTTCCAAGAGATAAGCTGACACTGGGCAAGCCCCTGGGAGAAGG TTGCTTTGGGCAAGTGGTCATGGCGGAAGCAGTGGGAATTGACAAAGACAAGCCCAAGGA GGCGGTCACCGTGGCCGTGAAGATGTTGAAAGATGATGCCACAGAGAAAGACCTTTCTGA TCTGGTGTCAGAGATGGAGATGATGAAGATGATTGGGAAACACAAGAATATCATAAATCT TCTTGGAGCCTGCACACAGGATGGGCCTCTCTATGTCATAGTTGAGTATGCCTCTAAAGG CAACCTCCGAGAATACCTCCGAGCCCGGAGGCCACCCGGGATGGAGTACTCCTATGACAT TAACCGTGTTCCTGAGGAGCAGATGACCTTCAAGGACTTGGTGTCATGCACCTACCAGCT GGCCAGAGGCATGGAGTACTTGGCTTCCCAAAAATGTATTCATCGAGATTTAGCAGCCAG AAATGTTTTGGTAACAGAAAACAATGTGATGAAAATAGCAGACTTTGGACTCGCCAGAGA TATCAACAATATAGACTATTACAAAAAGACCACCAATGGGCGGCTTCCAGTCAAGTGGAT GGCTCCAGAAGCCCTGTTTGATAGAGTATACACTCATCAGAGTGATGTCTGGTCCTTCGG GGTGTTAATGTGGGAGATCTTCACTTTAGGGGGCTCGCCCTACCCAGGGATTCCCGTGGA GGAACTTTTTAAGCTGCTGAAGGAAGGACACAGAATGGATAAGCCAGCCAACTGCACCAA CGAACTGTACATGATGATGAGGGACTGTTGGCATGCAGTGCCCTCCCAGAGACCAACGTT CAAGCAGTTGGTAGAAGACTTGGATCGAATTCTCACTCTCACAACCAATGAGCAAGCCAG GGCTGAGCAGGAAGAAGAATTCATTAGTAACACTTTATTCAAGAAAATTCAGGCTTTGCA GAAGGAGAAAGAAACCCTTGCTGTAAATTATGAGAAAGAAGAAGAATTCCTCACTAATGA GCTCTCCAGAAAATTGATGCAGTTGCAGCATGAGAAAGCCGAACTAGAACAGCATCTTGA ACAAGAGCAGGAATTTCAGGTCAACAAACTGATGAAGAAAATTAAAAAACTGGAGAATGA CACCATTTCTAAGCAACTTACATTAGAACAGTTGAGACGGGAGAAGATTGACCTTGAAAA TACATTGGAACAAGAACAAGAAGCACTAGTTAATCGCCTCTGGAAAAGGATGGATAAGCT TGAAGCTGAAAAGCGAATCCTGCAGGAAAAATTAGACCAGCCCGTCTCTGCTCCACCATC GCCTAGAGATATCTCCATGGAGATTGATTCTCCAGAAAATATGATGCGTCACATCAGGTT TTTAAAGAATGAAGTGGAACGGCTGAAGAAGCAACTGAGAGCTGCTCAGTTACAGCATTC AGAGAAAATGGCACAGTATCTGGAGGAGGAACGTCACATGAGAGAAGAGAACTTGAGGCT CCAGAGGAAGCTGCAGAGGGAGATGGAGAGAAGAGAAGCCCTCTGTCGACAGCTCTCCGA GAGTGAGTCCAGCTTAGAAATGGACGACGAAAGGTATTTTAATGAGATGTCTGCACAAGG ATTAAGACCTCGCACTGTGTCCAGCCCGATCCCTTACACACCTTCTCCGAGTTCAAGCAG GCCTATATCACCTGGTCTATCATATGCAAGTCACACGGTTGGTTTCACGCCACCAACTTC ACTGACTAGAGCTGGAATGTCTTATTACAATTCCCCGGGTCTTCACGTGCAGCACATGGG AACATCCCATGGTATCACAAGGCCTTCACCACGGAGAAGCAACAGTCCTGACAAATTCAA ACGGCCCACGCCGCCTCCATCTCCCAACACACAGACCCCAGTCCAGCCACCTCCGCCTCC ACCTCCGCCACCCATGCAGCCCACGGTCCCCTCAGCAGCCACCTCGCAGCCTACTCCTTC GCAACATTCGGCGCACCCCTCCTCCCAGCC TTAAFGFR2:OFD1 >ATGGTCAGCTGGGGTCGTTTCATCTGCCT (5229 base pairs)GGTCGTGGTCACCATGGCAACCTTGTCCCT (SEQ ID NO: 64)GGCCCGGCCCTCCTTCAGTTTAGTTGAGGA TACCACATTAGAGCCAGAAGAGCCACCAACCAAATACCAAATCTCTCAACCAGAAGTGTA CGTGGCTGCGCCAGGGGAGTCGCTAGAGGTGCGCTGCCTGTTGAAAGATGCCGCCGTGAT CAGTTGGACTAAGGATGGGGTGCACTTGGGGCCCAACAATAGGACAGTGCTTATTGGGGA GTACTTGCAGATAAAGGGCGCCACGCCTAGAGACTCCGGCCTCTATGCTTGTACTGCCAG TAGGACTGTAGACAGTGAAACTTGGTACTTCATGGTGAATGTCACAGATGCCATCTCATC CGGAGATGATGAGGATGACACCGATGGTGCGGAAGATTTTGTCAGTGAGAACAGTAACAA CAAGAGAGCACCATACTGGACCAACACAGAAAAGATGGAAAAGCGGCTCCATGCTGTGCC TGCGGCCAACACTGTCAAGTTTCGCTGCCCAGCCGGGGGGAACCCAATGCCAACCATGCG GTGGCTGAAAAACGGGAAGGAGTTTAAGCAGGAGCATCGCATTGGAGGCTACAAGGTACG AAACCAGCACTGGAGCCTCATTATGGAAAGTGTGGTCCCATCTGACAAGGGAAATTATAC CTGTGTAGTGGAGAATGAATACGGGTCCATCAATCACACGTACCACCTGGATGTTGTGGA GCGATCGCCTCACCGGCCCATCCTCCAAGCCGGACTGCCGGCAAATGCCTCCACAGTGGT CGGAGGAGACGTAGAGTTTGTCTGCAAGGTTTACAGTGATGCCCAGCCCCACATCCAGTG GATCAAGCACGTGGAAAAGAACGGCAGTAAATACGGGCCCGACGGGCTGCCCTACCTCAA GGTTCTCAAGGCCGCCGGTGTTAACACCACGGACAAAGAGATTGAGGTTCTCTATATTCG GAATGTAACTTTTGAGGACGCTGGGGAATATACGTGCTTGGCGGGTAATTCTATTGGGAT ATCCTTTCACTCTGCATGGTTGACAGTTCTGCCAGCGCCTGGAAGAGAAAAGGAGATTAC AGCTTCCCCAGACTACCTGGAGATAGCCATTTACTGCATAGGGGTCTTCTTAATCGCCTG TATGGTGGTAACAGTCATCCTGTGCCGAATGAAGAACACGACCAAGAAGCCAGACTTCAG CAGCCAGCCGGCTGTGCACAAGCTGACCAAACGTATCCCCCTGCGGAGACAGGTAACAGT TTCGGCTGAGTCCAGCTCCTCCATGAACTCCAACACCCCGCTGGTGAGGATAACAACACG CCTCTCTTCAACGGCAGACACCCCCATGCTGGCAGGGGTCTCCGAGTATGAACTTCCAGA GGACCCAAAATGGGAGTTTCCAAGAGATAAGCTGACACTGGGCAAGCCCCTGGGAGAAGG TTGCTTTGGGCAAGTGGTCATGGCGGAAGCAGTGGGAATTGACAAAGACAAGCCCAAGGA GGCGGTCACCGTGGCCGTGAAGATGTTGAAAGATGATGCCACAGAGAAAGACCTTTCTGA TCTGGTGTCAGAGATGGAGATGATGAAGATGATTGGGAAACACAAGAATATCATAAATCT TCTTGGAGCCTGCACACAGGATGGGCCTCTCTATGTCATAGTTGAGTATGCCTCTAAAGG CAACCTCCGAGAATACCTCCGAGCCCGGAGGCCACCCGGGATGGAGTACTCCTATGACAT TAACCGTGTTCCTGAGGAGCAGATGACCTTCAAGGACTTGGTGTCATGCACCTACCAGCT GGCCAGAGGCATGGAGTACTTGGCTTCCCAAAAATGTATTCATCGAGATTTAGCAGCCAG AAATGTTTTGGTAACAGAAAACAATGTGATGAAAATAGCAGACTTTGGACTCGCCAGAGA TATCAACAATATAGACTATTACAAAAAGACCACCAATGGGCGGCTTCCAGTCAAGTGGAT GGCTCCAGAAGCCCTGTTTGATAGAGTATACACTCATCAGAGTGATGTCTGGTCCTTCGG GGTGTTAATGTGGGAGATCTTCACTTTAGGGGGCTCGCCCTACCCAGGGATTCCCGTGGA GGAACTTTTTAAGCTGCTGAAGGAAGGACACAGAATGGATAAGCCAGCCAACTGCACCAA CGAACTGTACATGATGATGAGGGACTGTTGGCATGCAGTGCCCTCCCAGAGACCAACGTT CAAGCAGTTGGTAGAAGACTTGGATCGAATTCTCACTCTCACAACCAATGAGACACAACT TCGAAACCAGCTAATTCATGAGTTGATGCACCCTGTATTGAGTGGAGAACTGCAGCCTCG GTCCATTTCAGTAGAAGGGAGCTCCCTCTTAATAGGCGCCTCTAACTCTTTAGTGGCAGA TCACTTACAAAGATGTGGCTATGAATATTCACTTTCTGTTTTCTTTCCAGAAAGTGGTTT GGCAAAAGAAAAGGTATTTACTATGCAGGATCTATTACAACTCATTAAAATCAACCCTAC TTCCAGTCTCTACAAATCACTGGTTTCAGGATCTGATAAAGAAAATCAAAAAGGTTTTCT TATGCATTTTTTAAAAGAATTGGCAGAATATCATCAAGCTAAAGAGAGTTGTAATATGGA AACTCAGACAAGTTCGACATTTAACAGAGATTCTCTGGCTGAGAAGCTTCAGCTTATTGA TGATCAGTTTGCAGATGCTTACCCTCAGCGTATCAAGTTCGAATCTTTAGAAATAAAGCT AAATGAGTATAAGAGAGAAATAGAAGAGCAACTTCGGGCAGAAATGTGTCAAAAGTTGAA GTTTTTTAAAGATACCGAGATAGCAAAAATTAAAATGGAAGCAAAAAAAAAGTATGAAAA GGAGTTAACCATGTTCCAGAATGATTTTGAAAAAGCTTGTCAAGCAAAATCTGAAGCTCT CGTTCTTCGGGAAAAGAGTACCCTTGAAAGAATTCACAAGCACCAAGAGATTGAAACAAA AGAAATTTATGCTCAAAGGCAACTTTTACTAAAAGATATGGATTTGCTAAGAGGAAGAGA AGCAGAGCTGAAGCAAAGAGTTGAAGCTTTTGAATTGAACCAGAAGCTCCAGGAAGAAAA ACATAAAAGCATAACTGAGGCACTTAGGAGACAGGAGCAGAATATAAAGAGTTTTGAGGA GACCTATGACCGAAAGCTCAAGAATGAACTTCTAAAGTATCAACTTGAACTGAAGGATGA CTACATCATTAGAACTAATCGACTGATTGAAGATGAAAGGAAGAATAAAGAAAAAGCTGT TCATTTGCAAGAGGAGCTCATAGCTATTAATTCAAAAAAGGAGGAACTCAATCAATCTGT AAATCGTGTGAAAGAACTTGAGCTTGAATTAGAGTCTGTCAAAGCCCAGTCTTTGGCAAT AACAAAACAAAACCATATGCTGAATGAAAAGGTTAAAGAGATGAGTGATTATTCACTACT AAAAGAAGAGAAACTGGAGCTTCTGGCACAAAATAAATTACTTAAACAACAACTGGAAGA GAGTAGAAATGAAAACCTGCGTCTCCTAAACCGCCTAGCTCAGCCGGCTCCTGAACTTGC AGTCTTTCAGAAAGAACTACGGAAAGCCGAAAAGGCTATAGTGGTTGAGCATGAGGAGTT CGAAAGCTGCAGGCAAGCTCTGCACAAACAACTGCAAGACGAAATTGAGCATTCTGCACA GCTGAAGGCCCAGATTCTAGGTTACAAAGCTTCTGTAAAGAGTTTAACTACTCAGGTTGC CGATTTAAAATTGCAACTGAAGCAAACTCAGACAGCCCTAGAGAATGAAGTGTACTGCAA TCCAAAGCAGTCTGTGATCGATCGTTCTGTCAATGGATTAATAAATGGCAATGTGGTGCC TTGCAATGGTGAGATAAGTGGGGATTTCTTGAACAATCCTTTTAAACAGGAAAACGTTCT AGCACGTATGGTTGCATCAAGGATCACAAATTATCCAACTGCATGGGTGGAGGGTAGTTC CCCTGATTCTGACCTTGAGTTTGTAGCCAATACTAAGGCAAGGGTCAAAGAGCTTCAGCA AGAGGCCGAACGCTTGGAAAAGGCTTTCAGAAGTTACCATCGGAGAGTCATTAAAAACTC TGCCAAAAGCCCACTAGCAGCAAAGAGCCCACCATCTCTGCACTTGCTGGAAGCCTTCAA AAACATTACTTCCAGTTCCCCGGAAAGACATATTTTTGGAGAGGACAGAGTTGTCTCTGA GCAGCCTCAAGTGGGCACACTTGAAGAAAGGAATGACGTCGTGGAAGCACTGACAGGCAG TGCAGCCTCGAGGCTCCGCGGGGGCACTTCCTCCAGACGCCTCTCTTCCACACCCCTTCC AAAAGCAAAAAGAAGCCTCGAAAGTGAAATGTATCTGGAAGGTCTGGGCAGATCACACAT TGCTTCCCCCAGTCCTTGTCCTGACAGAATGCCCCTACCATCACCCACTGAGTCTAGGCA CAGCCTCTCCATCCCTCCTGTCTCCAGCCCTCCGGAGCAGAAAGTGGGTCTTTATCGAAG ACAAACTGAACTTCAAGACAAAAGTGAATTTTCAGATGTGGACAAGCTAGCTTTTAAGGA TAATGAGGAGTTTGAATCATCTTTTGAATCTGCAGGGAACATGCCAAGGCAGTTGGAAAT GGGCGGGCTTTCTCCTGCCGGGGATATGTCTCATGTGGACGCTGCTGCAGCTGCTGTGCC CCTCTCATATCAGCACCCAAGTGTAGATCAGAAACAAATTGAAGAACAAAAGGAAGAAGA AAAAATACGGGAACAGCAAGTGAAAGAACGAAGGCAGAGAGAAGAAAGAAGGCAGAGTAA CCTACAAGAAGTTTTAGAAAGGGAACGAAGAGAACTAGAAAAACTGTATCAGGAAAGGAA GATGATTGAAGAATCACTGAAGATTAAAATAAAAAAGGAATTAGAAATGGAAAATGAATT AGAAATGAGTAATCAAGAAATAAAAGACAAATCTGCTCACAGTGAAAATCCTTTAGAGAA ATACATGAAAATCATCCAGCAGGAGCAAGACCAGGAGTCGGCAGATAAGAGCTCAAAAAA GATGGTCCAAGAAGGCTCCCTAGTGGACACGCTGCAATCTAGTGACAAAGTCGAAAGTTT AACAGGCTTTTCTCATGAAGAACTAGACGA CTCTTGGTAA

What is claimed:
 1. A method of identifying a cancer patient that is responsive to treatment with a fibroblast growth factor receptor (FGFR) inhibitor comprising: evaluating a biological sample from the patient for a FGFR mutant from a FGFR mutant gene panel, wherein the FGFR mutant is a FGFR fusion gene or a FGFR single nucleotide polymorphism, and wherein said evaluating comprises amplifying cDNA with a pair of primers that bind to and amplify one or more FGFR mutants from the FGFR mutant gene panel; and determining whether the one or more FGFR mutants from the FGFR mutant gene panel are present in the sample, wherein the presence of the one or more FGFR mutants indicates that the patient is responsive to treatment with the FGFR inhibitor.
 2. The method of claim 1, wherein the FGFR fusion gene comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, or FGFR2:OFD1, or any combination thereof.
 3. The method of claim 1, wherein the FGFR single nucleotide polymorphism comprises R248C, S249C, G370C, or Y373C, or any combination thereof.
 4. The method of claim 1, wherein the cancer is bladder cancer and the FGFR mutant gene panel comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
 5. The method of claim 1, wherein the cancer is metastatic bladder cancer and the FGFR mutant gene panel comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
 6. The method of claim 1, wherein the cancer is ovarian cancer and the FGFR mutant gene panel comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
 7. The method of claim 1, wherein the cancer is head and neck cancer and the FGFR mutant gene panel comprises FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
 8. The method of claim 1, wherein the cancer is metastatic head and neck cancer and the FGFR mutant gene panel comprises FGFR3:BAIAP2L1, FGFR2:CASP7, or FGFR2:OFD1, or any combination thereof.
 9. The method of claim 1, wherein the cancer is esophageal cancer and the FGFR mutant gene panel comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR2:BICC1, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
 10. The method of claim 1, wherein the cancer is metastatic esophageal cancer and the FGFR mutant gene panel comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof.
 11. The method of claim 1, wherein the cancer is non-small-cell lung carcinoma adenocarcinoma and the FGFR mutant gene panel comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:AFF3, FGFR2:CASP7, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
 12. The method of claim 1, wherein the cancer is non-small cell lung carcinoma squamous cell carcinoma and the FGFR mutant gene panel comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
 13. The method of claim 1, wherein the cancer is metastatic endometrial cancer and the FGFR mutant gene panel comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:CASP7, FGFR2:CCDC6, or FGFR2:OFD1, or any combination thereof.
 14. The method of claim 1, wherein the cancer is breast cancer and the FGFR mutant gene panel comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCD6, or FGFR2:OFD1, or any combination thereof.
 15. The method of claim 1, wherein the cancer is hepatocellular carcinoma and the FGFR mutant gene panel comprises FGFR3:TACC3 v1, FGFR3:TACC3 v3, FGFR3:TACC3 Intron, FGFR3:BAIAP2L1, FGFR2:BICC1, FGFR2:AFF3, FGFR2:CASP7, FGFR2:CCDC6, FGFR2:OFD1, FGFR3 R248C, FGFR3 S249C, FGFR3 G370C, or FGFR3 Y373C, or any combination thereof.
 16. The method of claim 1, wherein the FGFR mutant and pair of primers are: FGFR3:TACC3 v1 and primers having the amino acid sequences of SEQ ID NO:5 and SEQ ID NO:6; FGFR3:TACC3 v3 and primers having the amino acid sequences of SEQ ID NO:7 and SEQ ID NO:8; FGFR3:TACC3 Intron and primers having the amino acid sequences of SEQ ID NO:9 and SEQ ID NO:10; FGFR3:BAIAP2L1 and primers having the amino acid sequences of SEQ ID NO:11 and SEQ ID NO:12; FGFR2:BICC1 and primers having the amino acid sequences of SEQ ID NO:13 and SEQ ID NO:14; FGFR2:AFF3 and primers having the amino acid sequences of SEQ ID NO:15 and SEQ ID NO:16; FGFR2:CASP7 and primers having the amino acid sequences of SEQ ID NO:17 and SEQ ID NO:18; FGFR2:CCDC6 and primers having the amino acid sequences of SEQ ID NO:19 and SEQ ID NO:20; FGFR2:OFD1 and primers having the amino acid sequences of SEQ ID NO:21 and SEQ ID NO:22; R248C and primers having the amino acid sequences of SEQ ID NO:23 and SEQ ID NO:24 or SEQ ID NO:31 and SEQ ID NO:32; S249C and primers having the amino acid sequences of SEQ ID NO:25 and SEQ ID NO:26 or SEQ ID NO:33 and SEQ ID NO:34; G370C and primers having the amino acid sequences of SEQ ID NO:27 and SEQ ID NO:28 or SEQ ID NO:35 and SEQ ID NO:36; Y373C and primers having the amino acid sequences of SEQ ID NO:29 and SEQ ID NO:30 or SEQ ID NO:37 and SEQ ID NO:38; or any combination thereof.
 17. The method of claim 1, wherein the evaluating comprises: isolating RNA from the biological sample and synthesizing cDNA from the isolated RNA.
 18. The method of claim 17, further comprising pre-amplifying the cDNA prior to the amplifying step.
 19. The method of claim 1, wherein the cDNA is preamplified.
 20. The method of claim 1, wherein the amplifying step comprises performing real-time PCR.
 21. The method of claim 20, wherein the real-time PCR is performed with one or more probes comprising SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, and/or SEQ ID NO:55.
 22. The method of claim 21, wherein the real-time PCR is further performed with one or more 3′ blocking oligonucleotides comprising SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, and/or SEQ ID NO:42.
 23. The method of claim 1, wherein said determining step comprises sequencing the amplified cDNA.
 24. A kit for identifying the presence of one or more FGFR mutant genes in a biological sample comprising: pairs of primers having the sequences of SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, or any combination thereof; and instructions for performing an assay to detect one or more FGFR mutant genes.
 25. The kit of claim 24, further comprising one or more probes, one or more 3′ blocking oligonucleotides, or both.
 26. The kit of claim 25, wherein a. the pair of primers have the sequences SEQ ID NO:5 and SEQ ID NO:6 and the probe has the sequence of SEQ ID NO:43; b. the pair of primers have the sequences SEQ ID NO:7 and SEQ ID NO:8 and the probe has the sequence of SEQ ID NO:44; c. the pair of primers have the sequences SEQ ID NO:9 and SEQ ID NO:10 and the probe has the sequence of SEQ ID NO:46; d. the pair of primers have the sequences SEQ ID NO:11 and SEQ ID NO:12 and the probe has the sequence of SEQ ID NO:47; e. the pair of primers have the sequences SEQ ID NO:13 and SEQ ID NO:14 and the probe has the sequence of SEQ ID NO:45; f. the pair of primers have the sequences SEQ ID NO:15 and SEQ ID NO:16 and the probe has the sequence of SEQ ID NO:48; g. the pair of primers have the sequences SEQ ID NO:17 and SEQ ID NO:18 and the probe has the sequence of SEQ ID NO:49; h. the pair of primers have the sequences SEQ ID NO:19 and SEQ ID NO:20 and the probe has the sequence of SEQ ID NO:50; i. the pair of primers have the sequences SEQ ID NO:21 and SEQ ID NO:22 and the probe has the sequence of SEQ ID NO:51; j. the pair of primers have the sequences SEQ ID NO:23 and SEQ ID NO:24 and the probe has the sequence of SEQ ID NO:52; k. the pair of primers have the sequences SEQ ID NO:25 and SEQ ID NO:26 and the probe has the sequence of SEQ ID NO:53; l. the pair of primers have the sequences SEQ ID NO:27 and SEQ ID NO:28 and the probe has the sequence of SEQ ID NO:54; m. the pair of primers have the sequences SEQ ID NO:29 and SEQ ID NO:30 and the probe has the sequence of SEQ ID NO:55; n. the pair of primers have the sequences SEQ ID NO:31 and SEQ ID NO:32, the probe has the sequence of SEQ ID NO:52, and the 3′ blocking oligonucleotide has the sequence of SEQ ID NO:39; o. the pair of primers have the sequences SEQ ID NO:33 and SEQ ID NO:34, the probe has the sequence of SEQ ID NO:53, and the 3′ blocking oligonucleotide has the sequence of SEQ ID NO:40; p. the pair of primers have the sequences SEQ ID NO:35 and SEQ ID NO:36, the probe has the sequence of SEQ ID NO:54, and the 3′ blocking oligonucleotide has the sequence of SEQ ID NO:41; q. the pair of primers have the sequences SEQ ID NO:37 and SEQ ID NO:38, the probe has the sequence of SEQ ID NO:55, and the 3′ blocking oligonucleotide has the sequence of SEQ ID NO:42; or r. any combination thereof.
 27. A primer having the amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 or any combination thereof.
 28. A set of primers having the sequences of SEQ ID NO:5 and SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:25 and SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, or any combination thereof.
 29. An oligonucleotide probe having the sequence of any one of SEQ ID NOs:43-55, or any combination thereof.
 30. An oligonucleotide having the sequence of any one of SEQ ID NOs:39-42, or any combination thereof. 